High-speed fluorescence image-enabled cell sorting
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA705647 | ENA |
REPOSITORIES: ENA
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Project description:Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. We address this by establishing high speed image-enabled cell sorting (ICS), which records multicolor fluorescence images, and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We combine ICS with CRISPR-pooled screens to identify novel regulators of the NF-κB pathway, enabling the completion of genome-wide image- based screens in around nine hours of run-time.
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Project description:Dopamine transmission is a monoaminergic system involved in reward processing and motor control. Volume transmission is thought to be the main mechanism by which monoamines modulate effector transmission though synaptic structures are scarcely described. In the present work we aimed to unravel the cellular and molecular synaptome of single projection pathways (Zhu F, Cizeron M, Qiu Z, Benavides-Piccione R, Kopanitsa MV, Skene NG, Koniaris B, DeFelipe J, Fransén E, Komiyama NH & Grant SGN (2018) Architecture of the Mouse Brain Synaptome. Neuron 99: 781–799.e10). To that end, we established a workflow combining fluorescence tracing of the dopaminergic pathway, fluorescence activated synaptosome sorting and mass spectrometry-based proteomics. With this approach we provide the first ex-vivo model to thoroughly analyse the cellular and molecular organisation of dopaminergic synapses from mouse striatum.
2022-06-09 | PXD027534 | Pride
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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: Not available
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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:Juvenile Fluorescence Raw sequence reads
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Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression. The whole experiment was carried out in triplicate, with two time points (5 and 7 days post-inoculation) and three cell types (pathogen-proximal, pathogen-distal and uninfected control), totalling 18 microarrays. Transgenic Arabidopsis thaliana Col-0 with the transgene pDMR6:GFP was used for all experiments. Seedling populations were inoculated with 30,000-60,000 spores/ml of Hyaloperonospora arabidopsis strain Noks1 at 7 days old, and overground tissue sampled at 5 and 7 days post-inoculation. Protoplasts were generated from these samples according to Grønlund et al. 2012 JOVE, and sorted by fluorescence activated cell sorting into a GFP-positive (pathogen-proximal) and GFP-negative (pathogen-distal) population. As a control, uninfected seedlings were also sampled at 12 and 14 days old (equivalent age of 5 and 7 days post-inoculation), protoplasts generated and sorted to yield a GFP-negative population of uninfected control cells. Gene expression was analysed using NimbleGen 12 x 135K microarrays, designed for the TAIR10 genome. Data was normalised using the Robust Multichip Averaging algorithm.
2015-07-13 | E-GEOD-67100 | biostudies-arrayexpress
Project description:Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 106 cells or M-bM-^HM-<1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing M-bM-^HM-<10% and M-bM-^HM-<1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low. The genomic DNA (gDNA) of each culture or sorted and amplifed DNA was analyzed in triplicate.
2015-01-31 | E-GEOD-54040 | biostudies-arrayexpress
Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression.
2015-07-13 | GSE67100 | GEO