Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting.
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ABSTRACT:
SUBMITTER: Nicolas-Avila JA
PROVIDER: S-EPMC7811054 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundGeneralized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians.MethodsUsing fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay.ResultsWe found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker.ConclusionsIn this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.
| S-EPMC5575905 | biostudies-literature
Project description:Novel fluorescence-activated cell sorting (FACS) strategies to prospectively purify functionally distinct cell populations from the human myofiber-associated (hMFA) cell compartment, including human Skeletal Muscle Precursor cells (hSMPs): HSMPs, identified as CD45-Mac1-GlyA-CD31-CD34-CD56intITGA7hi hMFA cells, are highly enriched for cells expressing the satellite cell marker PAX7 and show efficient myogenic and lack adipogenic capacity. CD45-CD11b-GlyA-CD31-CD34+ hMFA cells (CD34+ cells) do not express PAX7, lack myogenic and exhibit adipogenic activity. We used Affymetrix Human Genome U133 Plus 2.0 microarrays to gain deeper insights into the molecular underpinnings functionally and phenotypically discrete human myofiber-associated cell subsets.
2014-01-08 | E-GEOD-44227 | biostudies-arrayexpress
Project description:Novel fluorescence-activated cell sorting (FACS) strategies to prospectively purify functionally distinct cell populations from the human myofiber-associated (hMFA) cell compartment, including human Skeletal Muscle Precursor cells (hSMPs): HSMPs, identified as CD45-Mac1-GlyA-CD31-CD34-CD56intITGA7hi hMFA cells, are highly enriched for cells expressing the satellite cell marker PAX7 and show efficient myogenic and lack adipogenic capacity. CD45-CD11b-GlyA-CD31-CD34+ hMFA cells (CD34+ cells) do not express PAX7, lack myogenic and exhibit adipogenic activity.
2014-01-08 | GSE44227 | GEO
Project description:Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.
| S-EPMC5216387 | biostudies-literature
Project description:The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.
| S-EPMC4793971 | biostudies-literature
Project description:To date, there have been limited high quality libraries of cardiomyocyte maturation during the perinatal period, in part owing to the difficulty of isolating large perinatal cardiomyocytes. We previously developed a method utilizing large-particle fluorescent activated cell sorting (LP-FACS) to isolate adult cardiomyocytes for single cell RNA-seq (Kannan et al., Circ Res, 2019). We utilize this method to generate a reference of perinatal cardiomyocyte maturation.
2020-04-01 | GSE147807 | GEO
Project description:There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60,000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.
| S-EPMC4280329 | biostudies-literature
Project description:Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that - although the RNA isolated from these samples presented some degradation - it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis.
| S-EPMC3627819 | biostudies-literature
Project description:Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D) values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6)). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.
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Project description:Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ?2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.
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