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  • 1. Agostinho, Ana
    et al.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    van Schendel, Robin
    Hernandez-Hernandez, Abrahan
    Kouznetsova, Anna
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Höög, Christer
    High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation2016In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 17, no 6, p. 901-913Article in journal (Refereed)
    Abstract [en]

    During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super-resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild-type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister-chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.

  • 2.
    Akpe, Victor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Photophysical and Chemical Approaches to Cellular Biophysics2008Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The central theme in this thesis is reversibility. Two main attempts has been made to approach reversibility in cellular systems from both chemical and physical points of view. Reversibility of immunolabeling of proteins on the cell surface has been adressed by development of new fluorescent substances optimized for CALI (Chromophore-Assisted Laser Inactivation of protein). Aluminum phthalocyanine (AlPc) is here identified to be a good candidate for a new generation of fluorophores for efficient hydroxyl radical generation. It is shown that cells can be reversibly labeled with antibody-AlPc conjugates. In experiments on living cells the AlPcs were not only active as classic fluorophores but also as photocatalytic substances with destaining properties. Reversibility of cell immobilization is also reported, where cells cultured in microstructures were immobilized and 3D supported using hydrogels. Hydrogel formulation and application was optimized to achieve a system where both viability and ease of use was satisfied. Gel reversibility was actualized with pH and enzyme treatment. The developped method offers the possibility of stop flow culturing cells in controlled and reusable 3D environments.

  • 3.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nyokong, Tebello
    Osadebe, P. O.
    Photophysical and photochemical parameters of octakis (benzylthio) phthalocyaninato zinc, aluminium and tin: Red shift index concept in solvent effect on the ground state absorption of zinc phthalocyanine derivatives2010In: Journal of Molecular Structure, ISSN 0022-2860, E-ISSN 1872-8014, Vol. 984, no 1-3, p. 1-14Article in journal (Refereed)
    Abstract [en]

    This paper addresses the synthesis of octa-substituted benzylthio metallophthalocyanines (OBTMPcs) that contain the central metal ions of Zn2+, Al3+ and Sn4+. The ground state absorption of ZnPc(SR)(8) (OBTZnPc) along with the ZnPc derivatives, well documented in literature were used to study a new concept called the red shift index (RsI). The concept is based on the empirical values of RsI of the different complexes in solvent media. Unequivocally, parameters used in this paper show strong correlations that are consistent with the results obtained. For instance, 12,1 of the complexes tend to increase as the refractive index, n(D), and solvent donor, DN, of solvent increases. Photodegradation (photobleaching) quantum yield, phi(d) measurements of these compounds show that they are highly photostable, phi(d) (0.03-0.33 x 10(-5)). The triplet quantum yield, phi(T) (0.40-0.53) and the triplet lifetime, tau(T) (610-810 mu s) are within the typical range for metallophthalocyanines in DMSO. The photosensitisation efficiency. S-Delta, is relatively high for all the molecules (0.74-0.90). (C) 2010 Elsevier B.V. All rights reserved.

  • 4.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nyokong, Tebello
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Photophysics and photochemistry of zinc, aluminium and tin octakis (benzylthio) phthalocyanines2008Report (Other academic)
  • 5.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ogunsipe, Abimbola
    Madu, Christian
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Red-Shift Index Concept in Solvent Effects of Chromophore-Substituted Metallophthalocyanines: A Look at the Empirical Relationship of the Macroscopic Properties of the Solute-Solvent Interactions2015In: Journal of Solution Chemistry, ISSN 0095-9782, E-ISSN 1572-8927, Vol. 44, no 2, p. 307-326Article in journal (Refereed)
    Abstract [en]

    Solvent effects on the UV/vis spectra of metallopthalocyanines (MPcs) have been interpreted using the red-shift index concept (R (s) I). The concept connects empirically, direct, experimental, easily accessible optical spectral data, which are explained by considering the differential behavior of the solute-solvent interactions at the ground state and excited state using the spectral values of MPcs along with the derived concept, called the associated solvation energy (ASE). R (s) I is formulated from three fundamental parameters, which are: ground state electronic absorption spectrum, polarization red-shift and a scaling factor of MPc (N (dye)) in the respective solvents. The R (s) I is a reflection of the index value of the chromophore substituent of MPc in the solvent; thus, the concept can be used as a solvatochromic parameter to study a wide range of supramolecular and heterocyclic compounds that can be modified at their periphery or 'handles'. Particularly, in this study, the concept has been used to rank MPc candidates by using the statistical mean performance of the solvatochromic parameters, which are red shift index, polarizability efficiency and ASE. We hereby review the solvent effects on the UV/vis spectra of substituted and unsubstituted MPcs.

  • 6.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Characterization studies of aluminum phthalocyanine binding to antibodies from SKBR 3 cell line2008Report (Other academic)
  • 7.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Protein Technology.
    Madu, Christian
    Obirai, Joseph C.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Understanding the Photochemical Pathway of In Vitro Target Delivery of Aluminium Phthalocyanine: A Mechanistic Approach Using Radical Reaction Chemistry2014In: ChemPlusChem, ISSN 2192-6506, Vol. 79, no 5, p. 671-679Article in journal (Refereed)
    Abstract [en]

    A classical dye, aluminium phthalocyanine (AlPc), is used to study the photochemical processes involved in the chromophore-assisted laser inactivation technique. Both cell-free and cell-based systems are investigated by novel methods and radical reaction chemistry. Findings on the photochemical pathways in two models representing cell-free and a cell-based systems are reported. In the cell-free system, the unsubstituted, free, fluorescence-active photosensitiser AlPc recovers its fluorescence signal by means of phosphorescence through a reversible photobleaching process. In the cell-based system, photoactivation of substituted AlPc conjugated to an antibody results in the loss of fluorescence signal at the area examined. Reinjection of the AlPc-conjugated antibodies restores the fluorescence signal.

  • 8. Aperia, Anita Chatarina
    et al.
    Akkuratov, Evgeny E
    Fontana, Jacopo Maria
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Na+, K+-ATPase, a new class of plasma membrane receptors2016In: American Journal of Physiology - Cell Physiology, ISSN 0363-6143, E-ISSN 1522-1563, Vol. 310, no 7, p. C491-C495Article in journal (Refereed)
    Abstract [en]

    The Na(+), K(+)-ATPase (NKA) differs from most other ion transporters not only in its capacity to maintain a steep electrochemical gradient across the plasma membrane but also as a receptor for a family of cardiotonic steroids, to which ouabain belongs. Studies from many groups, performed during the last fifteen years, have demonstrated that ouabain, a member of the cardiotonic steroid family, can activate a network of signaling molecules and that NKA will also serve as a signal transducer that can provide a feed back loop between NKA and the mitochondria. This brief review summarizes the current knowledge and controversies with regard to the understanding of NKA signaling.

  • 9.
    Ardabili, Sahar
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Dean flow-coupled inertial focusing for ultra-high-throughput particle filtration2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 3, 2010, p. 1586-1588Conference paper (Refereed)
    Abstract [en]

    Particle manipulation represents an important and fundamental step prior to counting, sorting and detecting bio-particles. In this study, we report dean-coupled inertial focusing of particles in flows through a single curve microchannel at extremely high channel Reynold numbers (∼325). We found the lateral particle focusing position, xf to be fixed and largely independent of radius of curvature and whether particles are pre-focused (at equilibrium) entering the curvature or randomly distributed. Finally, using a single inlet, u-shaped, microchannel we demonstrate filtration of 10μm particles from 2 μm particles at throughputs several orders of magnitude higher than previously shown.

  • 10. Azarias, Guillaume
    et al.
    Kruusmagi, Markus
    Connor, Siobhan
    Akkuratov, Evgeny E.
    Liu, Xiao-Li
    Lyons, David
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Broberger, Christian
    Aperia, Anita
    A Specific and Essential Role for Na,K-ATPase alpha 3 in Neurons Co-expressing alpha 1 and alpha 32013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 4, p. 2734-2743Article in journal (Refereed)
    Abstract [en]

    Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous alpha 1, and the more selectively expressed alpha 3. Although neurological syndromes are associated with alpha 3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of alpha 3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of alpha 3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration ([Na+](i)) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+](i), similar to what could be expected following suprathreshold neuronal activity, selective inhibition of alpha 3 almost completely abolished the capacity to restore [Na+](i) in soma and dendrite. Recordings of Na, K-ATPase specific current supported the notion that when [Na+](i) is elevated in the neuron, alpha 3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of alpha 3 function in the central nervous system. The alpha isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the alpha 3-dependent current and restoration of dendritic [Na+](i) were significantly attenuated, indicating that alpha 3 is a target for phosphorylation and may participate in short term regulation of neuronal function.

  • 11.
    Barbe, Laurent
    et al.
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Oksvold, Per
    KTH, School of Biotechnology (BIO), Proteomics.
    Stenius, Anna
    KTH, School of Biotechnology (BIO).
    Lewin, Erland
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Björling, Erik
    KTH, School of Biotechnology (BIO).
    Asplund, Anna
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Pontén, Fredrik
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics.
    Toward a confocal subcellular atlas of the human proteome2008In: Molecular and cellular proteomics, ISSN 1535-9476, Vol. 7, no 3, p. 499-508Article in journal (Refereed)
    Abstract [en]

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

  • 12. Benninger, Richard K. P.
    et al.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Young, Stephen
    Taner, Sabrina B.
    Culley, Fiona I.
    Schnyder, Tim
    Neil, Mark A. A.
    Wuestner, Daniel
    French, Paul M. W.
    Davis, Daniel M.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 96, no 2, p. L13-L15Article in journal (Refereed)
    Abstract [en]

    We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.

  • 13.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bates, Mark
    Nanoscopy-imaging life at the nanoscale: a Nobel Prize achievement with a bright future2015In: Physica Scripta, ISSN 0031-8949, E-ISSN 1402-4896, Vol. 90, no 10, article id 108010Article in journal (Refereed)
    Abstract [en]

    A grand scientific prize was awarded last year to three pioneering scientists, for their discovery and development of molecular 'ON-OFF' switching which, when combined with optical imaging, can be used to see the previously invisible with light microscopy. The Royal Swedish Academy of Science announced on October 8th their decision and explained that this achievement-rooted in physics and applied in biology and medicine-was awarded with the Nobel Prize in Chemistry for controlling fluorescent molecules to create images of specimens smaller than anything previously observed with light. The story of how this noble switch in optical microscopy was achieved and how it was engineered to visualize life at the nanoscale is highlighted in this invited comment.

  • 14.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    STED microscopy: increased resolution for medical research?2014In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 276, no 6, p. 560-578Article, review/survey (Refereed)
    Abstract [en]

    Optical imaging is crucial for addressing fundamental problems in all areas of life science. With the use of confocal and two-photon fluorescence microscopy, complex dynamic structures and functions in a plethora of tissue and cell types have been visualized. However, the resolution of classical' optical imaging methods is poor due to the diffraction limit and does not allow resolution of the cellular microcosmos. On the other hand, the novel stimulated emission depletion (STED) microscopy technique, because of its targeted on/off-switching of fluorescence, is not hampered by a diffraction-limited resolution barrier. STED microscopy can therefore provide much sharper images, permitting nanoscale visualization by sequential imaging of individual-labelled biomolecules, which should allow previous findings to be reinvestigated and provide novel information. The aim of this review is to highlight promising developments in and applications of STED microscopy and their impact on unresolved issues in biomedical science.

  • 15.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ronnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Bondar, Alexander
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy2011In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 12, p. 16-Article in journal (Refereed)
    Abstract [en]

    Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

  • 16.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, L.
    Westin, L.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, A.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatial Distribution of DARPP-32 in Dendritic Spines2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e75155-Article in journal (Refereed)
    Abstract [en]

    The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

  • 17.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Rantanen, Ville
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy2012In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 75, no 2, p. 220-228Article in journal (Refereed)
    Abstract [en]

    Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.

  • 18.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    STED microscopy: towards broadened use and scope of applications2014In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 20, no 1, p. 127-133Article, review/survey (Refereed)
    Abstract [en]

    High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles. These simpler and cheaper realizations of STED are now becoming more broadly available. In parallel with the continuous development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can thus expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes.

  • 19. Blom, M.
    et al.
    Reis, K.
    Nehru, V.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gad, A. K. B.
    Aspenström, P.
    RhoD is a Golgi component with a role in anterograde protein transport from the ER to the plasma membrane2015In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 333, no 2, p. 208-219Article in journal (Refereed)
    Abstract [en]

    RhoD is a member of the Rho GTPase family and it coordinates actin dynamics and membrane trafficking. Activation of RhoD results in formation of filopodia, dissolution of stress fibers, and the subsequent formation of short actin bundles. In addition, RhoD localizes to early endosomes and recycling endosomes, and has a regulatory role in endosome trafficking. In this study, we report on a function of RhoD in the regulation of Golgi homeostasis. We show that manipulation of protein and activation levels of RhoD, as well as of its binding partner WHAMM, result in derailed localization of Golgi stacks. Moreover, vesicle trafficking from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus measured by the VSV-G protein is severely hampered by manipulation of RhoD or WHAMM. In summary, our studies demonstrate a novel role for this member of the Rho GTPases in the regulation of Golgi function.

  • 20. Bollampalli, V. P.
    et al.
    Harumi Yamashiro, L.
    Feng, X.
    Bierschenk, D.
    Gao, Y.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Henriques-Normark, B.
    Nylén, S.
    Rothfuchs, A. G.
    BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 10, article id e1005206Article in journal (Refereed)
    Abstract [en]

    The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

  • 21.
    Brismar, Hjalmar
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Scott, Lena
    Malmersjö, Seth
    Kowalewski, Jacob M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zettergren Markus, Eivor
    Ulfhake, Brun
    Nairn, Angus C.
    Greengard, Paul
    Aperia, Anita
    Kvinnor och barns hälsa, Karolinska Institutet.
    Laterally diffusing dopamine 1 receptors are recruited to dendrtic spines by interaction with allosterically changed NMDA receptors2005In: Neuroscience 2005, 2005Conference paper (Refereed)
  • 22. Burlaka, Ievgeniia
    et al.
    Liu, Xiao Li
    Rebetz, Johan
    Arvidsson, Ida
    Yang, Liping
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karpman, Diana
    Aperia, Anita
    Ouabain Protects against Shiga Toxin-Triggered Apoptosis by Reversing the Imbalance between Bax and Bcl-xL2013In: Journal of the American Society of Nephrology, ISSN 1046-6673, E-ISSN 1533-3450, Vol. 24, no 9, p. 1413-1423Article in journal (Refereed)
    Abstract [en]

    Hemolytic uremic syndrome, a life-threatening disease often accompanied by acute renal failure, usually occurs after gastrointestinal infection with Shiga toxin 2 (Stx2)-producing Escherichia coli. Stx2 binds to the glycosphingolipid globotriaosylceramide receptor, expressed by renal epithelial cells, and triggers apoptosis by activating the apoptotic factor Bax. Signaling via the ouabain/Na,K-ATPase/IP3R/NF-B pathway increases expression of Bcl-xL, an inhibitor of Bax, suggesting that ouabain might protect renal cells from Stx2-triggered apoptosis. Here, exposing rat proximal tubular cells to Stx2 in vitro resulted in massive apoptosis, upregulation of the apoptotic factor Bax, increased cleaved caspase-3, and downregulation of the survival factor Bcl-xL; co-incubation with ouabain prevented all of these effects. Ouabain activated the NF-B antiapoptotic subunit p65, and the inhibition of p65 DNA binding abolished the antiapoptotic effect of ouabain in Stx2-exposed tubular cells. Furthermore, in vivo, administration of ouabain reversed the imbalance between Bax and Bcl-xL in Stx2-treated mice. Taken together, these results suggest that ouabain can protect the kidney from the apoptotic effects of Stx2.

  • 23. Burlaka, Ievgeniia
    et al.
    Nilsson, Linnea M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Scott, Lena
    Holtback, Ulla
    Eklöf, Ann-Christine
    Fogo, Agnes B.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Aperia, Anita
    Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease2016In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 90, no 1, p. 135-148Article in journal (Refereed)
    Abstract [en]

    There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time-and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.

  • 24.
    Carrick, Christopher
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology.
    Larsson, Per A.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aidun, Cyrus
    Wågberg, Lars
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Native and functionalized micrometre-sized cellulose capsules prepared by microfluidic flow focusing2014In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 4, no 37, p. 19061-19067Article in journal (Refereed)
    Abstract [en]

    Cellulose capsules with average outer and inner radii of approximately 44 mu m and 29 mm respectively were prepared from cellulose dissolved in a mixture of lithium chloride and dimethylacetamide using a microfluidic flow focusing device (MFFD). The MFFD had three inlets where octane oil in a cellulose solution in silicone oil was used to produce a double emulsion containing a cellulose capsule. This technique enables the formation of capsules with a narrow size distribution which can be beneficial for drug delivery or controlled release capsules. In this respect, cellulose is a highly interesting material since it is known to cause no autoimmune reactions when used in contact with human tissue. Furthermore, by controlling the chemical properties of the cellulose, it is possible to trigger a swelling of the capsules and consequentially the release of an encapsulated substance, e. g. a model drug, when the capsule becomes exposed to an external stimulus. To demonstrate this, capsules were functionalized by carboxymethylation to be pH- responsive and to expand approximately 10% when subjected to a change in pH from 3 to 10. The diffusion constant of a model drug, a 4 kDa fluorescently labelled dextran, through the native capsule wall was estimated to be 6.5 X 10(-14) m(2) s(-1) by fitting fluorescence intensity data to Fick's second law.

  • 25. Chen, Yun
    et al.
    Molnar, Mátyás
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Li, Li
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Friberg, Peter
    Gan, Li-Ming
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Characterization of VCAM-1-Binding Peptide-Functionalized Quantum Dots for Molecular Imaging of Inflamed Endothelium2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 12, p. e83805-Article in journal (Refereed)
    Abstract [en]

    Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs) have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide) functionalized QDs (VQDs) from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor alpha (TNF-alpha) treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF-alpha-treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.

  • 26.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Khorshidi, Mohammad Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kadri, Nadir
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity2013In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 5, no 4, p. 712-719Article in journal (Refereed)
    Abstract [en]

    Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.

  • 27.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Solid tumor spheroid formation by temperature-controlled high voltage ultrasound in a multi-well microdevice2014In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems SocietyChemical and Biological Microsystems Society , 2014, p. 573-575Conference paper (Refereed)
    Abstract [en]

    In the present work we demonstrate effective 3D growth of human hepatocellular carcinoma (HCC) HepG2 cell spheroids in parallel in a multi-well microdevice actuated with high voltage ultrasound in a temperature-controlled system. We compare the spheroid formation during continuous ultrasound exposure for one week where we formed spheroids in 59% of the wells, with the spheroid formation without ultrasound actuation, where we obtained 0% spheroids. Furthermore, we present an application of the tumor spheroids for investigating natural killer (NK) cells behavior against solid tumors.

  • 28.
    Christakou, Athanasia E.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasonic three-dimensional cell culture on chip for dynamic studies of tumor immune surveillance by natural killer cellsManuscript (preprint) (Other academic)
  • 29.
    Christakou, Athanasia
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells2015In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, no 15, p. 3222-31Article in journal (Refereed)
    Abstract [en]

    We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

  • 30. Christensen, B M
    et al.
    Zelenina, M
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, A
    Nielsen, S
    Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.2000In: American journal of physiology. Renal physiology, ISSN 1931-857X, Vol. 278, no 1, p. F29-42Article in journal (Refereed)
    Abstract [en]

    Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.

  • 31. Clausson, Carl-Magnus
    et al.
    Arngarden, Linda
    Ishaq, Omer
    Klaesson, Axel
    Kuhnemund, Malte
    Grannas, Karin
    Koos, Bjorn
    Qian, Xiaoyan
    Ranefall, Petter
    Krzywkowski, Tomasz
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Mats
    Wahlby, Carolina
    Soderberg, Ola
    Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12317Article in journal (Refereed)
    Abstract [en]

    Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.

  • 32.
    Dånmark, Staffan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Gladnikoff, Micha
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Mustafa, Kamal
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Finne-Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Development of a novel microfluidic device for long-term in situ monitoring of live cells in 3-dimensional matrices2012In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 14, no 5, p. 885-893Article in journal (Refereed)
    Abstract [en]

    Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated. We have developed a microfluidic cell culture device that incorporates a biodegradable rigid 3D polymer scaffold using standard soft lithography methods. The device permits repeated high-resolution fluorescent imaging of live cell populations within the matrix over a 4 week period. It was also possible to track cell development at the same spatial location throughout this time. In addition, human primary periodontal ligament cells were induced to produce quantifiable calcium deposits within the system. This simple and versatile device should be readily applicable for cell-based studies that require long-term culture and high-resolution bioimaging.

  • 33.
    Dånmark, Staffan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Gladnikoff, Micha
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Mustafa, Kamal
    Insititutt for klinisk Odontologi, Medicinska och Odontologiska Fakulteten, Universitetet i Bergen, Norge.
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Finne-Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Development of Novel Microfluidic Device for Long-Term in situ Monitoring of Live Cells in 3-dimensional MatricesManuscript (preprint) (Other academic)
  • 34. Enqvist, M.
    et al.
    Ask, E. H.
    Forslund, E.
    Carlsten, M.
    Abrahamsen, G.
    Béziat, V.
    Andersson, S.
    Schaffer, M.
    Spurkland, A.
    Bryceson, Y.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, K. -J
    Coordinated expression of DNAM-1 and LFA-1 in educated NK cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 194, no 9, p. 4518-4527Article in journal (Refereed)
    Abstract [en]

    The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I-specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.

  • 35.
    Errando-Herranz, Carlos
    et al.
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Vastesson, Alexander
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Pardon, Gaspard
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Bergström, Gunnar
    Wijngaart, Wouter van der
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Haraldsson, Tommy
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Gylfason, Kristinn B.
    KTH, School of Electrical Engineering (EES), Micro and Nanosystems.
    Biocompatibility of OSTE polymers studied by cell growth experiments2013In: Proceedings of the 17th Int. Conf. on Miniaturized Systems for Chemistry  and Life Sciences (microTAS), Freiburg, Germany, 2013Conference paper (Refereed)
    Abstract [en]

    The recently introduced OSTE polymer technology has shown very useful features for microfluidics for lab-on-a-chip applications. However, no data has yet been published on cell viability on OSTE. In this work, we study the biocompatibility of three OSTE formulations by cell growth experiments. Moreover, we investigate the effect of varying thiol excess on cell viability on OSTE surfaces. The results show poor cell viability on one OSTE formulation, and viability comparable with polystyrene on a second formulation with thiol excess below 60%. In the third formulation, we observe cell proliferation. These results are promising for cell-based assays in OSTE microfluidic devices.

  • 36. Fenton, Robert A.
    et al.
    Moeller, Hanne B.
    Zelenina, Marina
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Snaebjornsson, Marteinn T.
    Holen, Torgeir
    MacAulay, Nanna
    Differential water permeability and regulation of three aquaporin 4 isoforms2010In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 67, no 5, p. 829-840Article in journal (Refereed)
    Abstract [en]

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K+ concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.

  • 37.
    Fontana, Jacopo
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fritz, Nicolas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, Anita
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Apoptosis caused by excessive mitochondrial albumin uptake in renal cells is initiated by increased mitochondrial calcium concentration2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29Article in journal (Other academic)
  • 38. Fontana, Jacopo M.
    et al.
    Burlaka, Ievgeniia
    Khodus, Georgiy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Calcium oscillations triggered by cardiotonic steroids2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, p. 5450-5455Article, review/survey (Refereed)
    Abstract [en]

    Na+, K+-ATPase (NKA) is well known for its function as an ion pump. Studies during the last decade have revealed an additional role for NKA as a signal transducer. In this brief review, we describe how cardiotonic steroids, which are highly specific NKA ligands, trigger slow Ca2+ oscillations by promoting the interaction between NKA and the inositol trisphosphate receptor, and how this Ca2+ signal activates the NF-B subunit p65 and increases the expression of the antiapoptotic factor Bcl-xL. The potential tissue-protective effects of this signal are discussed.

  • 39. Foo, K. S.
    et al.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Broberger, C.
    DISTRIBUTION AND NEUROPEPTIDE COEXISTENCE OF NUCLEOBINDIN-2 mRNA/NESFATIN-LIKE IMMUNOREACTIVITY IN THE RAT CNS2008In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 156, no 3, p. 563-579Article in journal (Refereed)
    Abstract [en]

    The protein fragment nesfatin-1 was recently implicated in the control of food intake. Central administration of this fragment results in anorexia and reduced body weight gain, whereas antisense or immunological nesfatin-1 antagonism causes increased food intake and overweight. Nesfatin-1 is derived from the precursor nucleobindin-2 (NUCB2). To identify the neurocircuitry underpinning the catabolic effects of NUCB2/nesfatin-1, we have used in situ hybridization and immunohistochemistry to map the distribution of this protein and its mRNA in the rat CNS and performed double-labeling experiments to localize its expression to functionally defined neuronal populations. These experiments confirm previous observations but also present several novel NUCB2 cell populations. Both NUCB2 mRNA and nesfatin-like immunoreactivity was most concentrated in the hypothalamus, in the supraoptic, paraventricular, periventricular and arcuate nuclei and the lateral hypothalamic area/perifornical region. Additionally, outside of the hypothalamus, labeling was observed in the thalamic parafascicular nucleus, the Edinger-Westphal nucleus, locus coeruleus, ventral raphe system, nucleus of solitary tract and in the preganglionic sympathetic intermediolateral cell column of the spinal cord, and the pituitary anterior and intermediate lobes. In neurons, immunoreactivity was almost exclusively confined to perikarya and primary dendrites with virtually no labeling of axonal terminals. Double-labeling immunohistochemistry revealed colocalization of nesfatin with vasopressin and oxytocin in magnocellular neuroendocrine neurons, thyrotropin-releasing hormone, corticotropin-releasing hormone, somatostatin, neurotensin, and growth-hormone-releasing hormone in parvocellular neuroendocrine neurons, pro-opiomelanocortin (but not neuropeptide Y) in the arcuate nucleus and melanin-concentrating hormone (but not hypocretin) in the lateral hypothalamus. Furthermore, nesfatin was extensively colocalized with cocaine- and amphetamine-regulated transcript in almost all NUCB2-expressing brain regions. These data reveal a wider distribution of NUCB2/nesfatin-1 than previously known, suggesting that the metabolic actions of this protein may involve not only feeding behavior but also endocrine and autonomic effects on energy expenditure. In addition, the subcellular distribution of nesfatin-like immunoreactivity indicates that this protein may not be processed like a conventional secreted neuromodulator.

  • 40. Forslund, E.
    et al.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Christakou, Athanasia E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations2012In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, no OCT, p. 300-Article in journal (Refereed)
    Abstract [en]

    Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.

  • 41. Forslund, Elin
    et al.
    Sohlberg, Ebba
    Enqvist, Monika
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmberg, Karl-Johan
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Sci Life Lab, Dept Microbiol Tumor & Cell Biol, S-17165 Stockholm, Sweden.
    Microchip-Based Single-Cell Imaging Reveals That CD56(dim) CD57(-)KIR(-)NKG2A(+) NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56(dim)CD57(-)KIR(-)NKG2A(-) NK Cells2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 195, no 7, p. 3374-3381Article in journal (Refereed)
    Abstract [en]

    NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(+) (lacking inhibitory receptors; IR-) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR-NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR- NK cells were generally more short-lived and IR- NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR- NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

  • 42.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 43.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nordberg, Erika
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Adams, Gregory P.
    Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor2007In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, p. 189-199Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents; for radionuclide based imaging applications in various carcinomas ils discussed.

  • 44. Frisk, T.
    et al.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherbergen, B.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Live-cell imaging of natural killer cell mediated tumor rejection in arrays of microwells2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 2, 2010, p. 950-952Conference paper (Refereed)
    Abstract [en]

    Due to the inherent heterogeneity of immune cell populations a comprehensive view of immune functions can only be achieved by collecting data from many individual cells. We here demonstrate a novel multi-well microchip platform for cell analysis, which we use to study the interaction between natural killer (NK) cells and their target cells.

  • 45.
    Frisk, Thomas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Khorshidi, Mohammad Ali
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution2011In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 13, no 4, p. 683-693Article in journal (Refereed)
    Abstract [en]

    We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.

  • 46.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Cultivation of COS7-cells using extracellular matrix in 3D microfluidic surface enlarged structure2005In: Micro Total Analysis Systems 2004, 2005, no 297, p. 261-263Conference paper (Refereed)
    Abstract [en]

    This paper presents a novel method to cultivate cells in a controlled 3D surface enlarged micro-environment. The 3D environment is achieved by insertion of a gel-like extracellular matrix (ECM) mixed with cells into a micromachined silicon fluid structure. Shrinking of the gel enables further flow through the channel. Due to the structure design the shrinking is non-uniform, which results in an increased surface area. With the proposed design cells are alive and viable after 72 h of incubation within the chip.

  • 47.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Three dimensional asymmetric microenvironment for cell biological studies2005In: Proceedings of µTAS 2005 Conference, 2005, p. 915-917Conference paper (Refereed)
    Abstract [en]

    We report on a method for three-dimensional cultivation of cells in a microstructured asymmetric environment. In the system an asymmetric environment is created by using diffusion through a gel of extra cellular matrix proteins surrounded by microfluidic flow channels. Individual cells embedded in the gel react on the concentration gradient. The system has been evaluated both for diffusion properties and based on the cellular response.

  • 48.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A concept for miniaturized 3-D cell culture using an extracellular matrix gel2005In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, no 24, p. 4751-4758Article in journal (Refereed)
    Abstract [en]

    This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.

  • 49.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Three dimensional asymmetric microenvironment for cell biologigal studies2005In: Proceedings Micro Total Analysis Systems (muTAS) 2005, 2005Conference paper (Refereed)
  • 50.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Liebmann, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A microfluidic device for parallel 3-D cell cultures in asymmetric environments2007In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 24, p. 4705-4712Article in journal (Refereed)
    Abstract [en]

    We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cello. Culture for up to two weeks was performed showing cells still viable and proliferating. The cell tracer dye calcein was used to verify gradient formation as the fluorescence intensity in exposed cells was proportional to the position in the chamber. Cellular response to an applied stimulus was demonstrated by use of an adenosine triphosphate gradient where the onset of a stimulated intracellular calcium release also depended on cell position.

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