| 1. |
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| 2. |
- Billecke, N, et al.
(författare)
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Perilipin 5 mediated lipid droplet remodelling revealed by coherent Raman imaging
- 2015
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Ingår i: Integrative biology : quantitative biosciences from nano to macro. - : Oxford University Press (OUP). - 1757-9708. ; 7:4, s. 467-476
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative, label-free coherent Raman microscopy was used to show lipid droplet compositional differences in muscle upon plin5 overexpressionin vivoandin vitro.
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| 3. |
- Castro Zalis, Marina, et al.
(författare)
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Label-free concentration of viable neurons, hESCs and cancer cells by means of acoustophoresis.
- 2016
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9708 .- 1757-9694. ; 8:3, s. 332-340
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Tidskriftsartikel (refereegranskat)abstract
- Concentration of viable cell populations in suspension is of interest for several clinical and pre-clinical applications. Here, we report that microfluidic acoustophoresis is an effective method to efficiently concentrate live and viable cells with high target purity without any need for protein fluorescent labeling using antibodies or over-expression. We explored the effect of the acoustic field acoustic energy density and systematically used different protocols to induce apoptosis or cell death and then determined the efficiency of live and dead cell separation. We used the breast cancer cell line MCF-7, the mouse neuroblastoma N2a as well as human embryonic stem cells (hESCs) to demonstrate that this method is gentle and can be applied to different cell populations. First, we induced cell death by means of high osmotic shock using a high concentration of PBS (10×), the protein kinase inhibitor staurosporine, high concentrations of dimethyl sulfoxide (DMSO, 10%), and finally, cell starvation. In all the methods employed, we successfully induced cell death and were able to purify and concentrate the remaining live cells using acoustophoresis. Importantly, the concentration of viable cells was not dependent on a specific cell type. Further, we demonstrate that different death inducing stimuli have different effects on the intrinsic cell properties and therefore affect the efficiency of the acoustophoretic separation.
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| 4. |
- Christakou, Athanasia E., et al.
(författare)
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Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
- 2013
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 5:4, s. 712-719
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Exploring DNA-protein interactions on the single DNA molecule level using nanofluidic tools
- 2017
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Ingår i: Integrative Biology (United Kingdom). - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 9:8, s. 650-661
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Tidskriftsartikel (refereegranskat)abstract
- DNA-protein interactions are at the core of the cellular machinery and single molecule methods have revolutionized the possibilities to study, and our understanding of these interactions on the molecular level. Nanofluidic channels have been extensively used for studying single DNA molecules during the last twelve years and in this review, we discuss how this experimental platform has been extended to studies of DNA-protein interactions. We first present how the design of the device can be tailored for the specific DNA-protein system studied and how the channels can be passivated to avoid non-specific binding of proteins. We then focus on describing the different kinds of DNA-interacting proteins that have been studied in nanofluidic devices, including proteins that compact DNA and proteins that form filaments on DNA. Our main objective is to highlight the diverse functionalitiesof DNA-protein systems that have been characterized using nanofluidic structures and hence demonstrate the versatility of these experimental tools. We finally discuss potential future directions studies of DNA-protein complexes in nanochannels might take, including specific DNA-protein systems that are difficult to analyze with traditional techniques, devices with increased complexity, and fully integrated lab-on-a-chip devices for analysis of material extracted from (single) cells.
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| 6. |
- Gilbert, Penney M., et al.
(författare)
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A single cell bioengineering approach to elucidate mechanisms of adult stem cell self-renewal
- 2012
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 4:4, s. 360-367
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Tidskriftsartikel (refereegranskat)abstract
- The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, elucidating the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal, is critical. Through careful design of bioengineered culture platforms, analysis of specific proteins presented to stem cells is possible. Critical to the success of the approach is single cell analysis, as more rapidly proliferating progenitors may mask the behavior of stem cells that proliferate slowly. Bioengineering approaches provide a potent means of gaining insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and the mechanisms that control their diverse fates. Ultimately, the multidisciplinary approach presented here will lead to novel therapeutic strategies for degenerative diseases.
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| 7. |
- Irimia, Daniel, et al.
(författare)
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Genome-wide transcriptome analysis of 150 cell samples
- 2009
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 1:1, s. 99-107
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Tidskriftsartikel (refereegranskat)abstract
- major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels, require nanogram amounts of total RNA. To overcome the sample size limitation of Current technologies, we have developed a method to probe the global gene expression ill biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by Stimulation, which are consistent with the information recognized in bulk-isolated samples.
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| 8. |
- Khorshidi, Mohammad Ali, et al.
(författare)
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Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro
- 2011
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 3:7, s. 770-778
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Tidskriftsartikel (refereegranskat)abstract
- We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.
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| 9. |
- Kiss, A, et al.
(författare)
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Non-monotonic cellular responses to heterogeneity in talin protein expression-level
- 2015
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Ingår i: Integrative biology : quantitative biosciences from nano to macro. - : Oxford University Press (OUP). - 1757-9708. ; 7:10, s. 1171-1185
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Tidskriftsartikel (refereegranskat)abstract
- Correlative imaging in single-cells of both live migration and post-fixation talin-labeling revealed non-monotonic correspondences between cellular properties and talin expression-levels.
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| 10. |
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