EdU labeling and visualization was done using Click-iT® EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific). Briefly, 1 × 105 NCC-CDS-X1 cells were plated in each well of a 24-well plate. The following day, continual treatment with 0.3 µM iP300w was initiated. For the pulse experiment, cells were treated with iP300w for 4 or 24 h and analyzed 24 h later. Cells were cultured with EdU (10 nM) in the last 4 h of the experiment, and samples were prepared for analyses according to the manufacture’s instruction. FACS analyses were performed on a BD FACSAria instrument and data was analyzed using FlowJo (BD Biosciences). Experiments were performed on at least three biological replicates.
Facsaria instrument
Manufactured by BD
Sourced in United States
The FACSAria is a cell sorter instrument designed for high-performance cell analysis and sorting. It enables the efficient separation and isolation of specific cell populations from complex samples. The FACSAria utilizes advanced fluidics and optics technology to provide accurate and reliable cell sorting capabilities.
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Lab products found in correlation
Lsrii flow cytometer, by BD (3 mentions)
Pspcas9 bb 2a gfp, by Addgene (2 mentions)
Cd45 percp cy5, by BioLegend (2 mentions)
Verapamil, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (2 mentions)
Facscalibur instrument, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Lipofectamine cas9 plus reagent, by Thermo Fisher Scientific (2 mentions)
Click it edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Accutase cell detachment solution, by Merck Group (1 mentions)
70 μm pre separation filters, by Miltenyi Biotec (1 mentions)
Fortessa analyser, by BD (1 mentions)
Apc cy7, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Liberase, by Roche (1 mentions)
Smart seq v4 ultra low input rna kit, by Takara Bio (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer, by Thermo Fisher Scientific (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
57 protocols using facsaria instrument
1
Cell Proliferation Dynamics by EdU Labeling
2
Isolation and Characterization of Fetal Gonadal Cells
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Fetal gonads were digested with an Accutase Cell Detachment Solution (Millipore, SCR005) at 37 °C for 5–15 min and the cell suspension was filtered through 70 μm Pre-Separation Filters (Miltenyi Biotec, 130-095-823; Supplementary information, Table S6 ). After centrifuging the cell suspension and discarding the supernatant, the cells were resuspended in L15 medium (plus 10% FBS). A PE-conjugated mouse anti-human C-KIT antibody (BD PharMingen, 555714) was used for fetal testis samples. A PE-conjugated mouse anti-human C-KIT (CD117) antibody (BD PharMingen, 555714), FITC-conjugated anti-human PECAM1 (CD31) antibody (Biolegend, 303104) and APC-conjugated anti-human IL13RA2 (CD213α2) antibody (Biolegend, 354406) were used for fetal ovary samples.7 (link) Cell sorting was performed using a BD FACSAria instrument (Special Order Research Product), and the data were analyzed using FlowJo software (Tree Star).
3
Detailed Workflow for Cell Sorting and Flow Cytometry
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For cell sorting: 30 × 10E6 cells were counted and resuspended in 300 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 15 μl of the human anti-CD19 antibody conjugated with BV510 (Becton Dickinson, 562947) and 15 μl of human anti-cd11b (Mac1) antibody conjugated with PE-Cy7 (eBioscience, 25-0118-41) were added. Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 2 ml of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were sorted in a BD FACS Aria instrument at the Flow Cytometry Unit of the Centre for Genomic Regulation.
For flow cytometry analysis : 1 × 10E6 cells were counted and resuspended in 100 μl PBS + 3% FBS in the presence of FcR blocking reagent. Cells were incubated for 10 min and 5 μl of each of the corresponding antibodies were added. For the CD19 knockout experiment, we used the antibody anti-CD19 conjugated with APC-Cy7 (Becton Dickinson, 557791). Cells were incubated for 30 min in the dark, washed with PBS and resuspended in 500 ul of PBS + 3% FBS. Topro-3 was added as a viability marker. Cells were measured in a BD Fortessa analyser. For the Stain Index calculation we used the formula: (mean positive—mean background) / (2 * SD background), as previously described [63 (link)].
Cell cytometry data is available in FlowRespository database (https://flowrepository.org ) [64 (link)].
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Cell cytometry data is available in FlowRespository database (
4
Zebrafish Endothelial Cell Isolation
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Zebrafish embryos were euthanized by tricaine overdose, minced with a razor blade in PBS, and digested in collagenase with agitation (Liberase, Roche). Endothelial cells were isolated after two rounds of cell sorting. In the first round, kdrl:EGFP(+) cells were enriched from total live cells. Enriched endothelial cells were resorted into kdrl:EGFP(+); lyve1b:dsRed(+) and kdrl:EGFP(+); lyve1b:dsRed(−) fractions. For kidney marrow FACS, adult zebrafish were euthanized by ice water immersion, kidney marrow was dissected and triturated ice-cold PBS. Erythrocytes were lysed with NH4Cl. For intracellular phosphoprotein staining, cells were fixed in PFA for 10 min, dehydrated in methanol, and washed before antibody staining in the presence of 0.5% BSA and 0.02% sodium azide. Sorting was performed on a BD FACSAria instrument and analysis was performed on a BD LSRII instrument.
5
CRISPR-Mediated PPP6C Knockout
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CRISPR/Cas9 constructs were generated by cloning sgRNA sequences into pSpCas9(BB)-2A-GFP (Addgene, 48138) according to the cloning protocol established by the Zhang lab (https://www.addgene.org/browse/article/7475/ ). Two sets of sgRNA oligos were used but only one sgRNA targeting exon 4 resulted in PPP6C knockout clones. pSpCas9(BB)-2A-GFP was used to generate negative control clones. 501mel cells were transfected with CRISPR/Cas9 constructs via PEI, and 48 hours post transfection, cells were trypsinized. After centrifugation and removal of media/trypsin, cells were resuspended in PBS and transferred to FACS tubes. Single GFP positive cells were sorted into 96 well plates via a BD FACSAria instrument. 96 well plates were treated with 0, 1, or 2 nM trametinib and incubated for several weeks until colonies were observed. PPP6C knockout 501mel cell colonies only grew out in the presence of trametinib and were maintained in 1–2 nM trametinib but withdrawn from trametinib ≥ 24 h before experiments. PPP6C knockout was confirmed by immunoblot and Sanger sequencing of PCR amplified target site.
6
Isolation and Sorting of Murine γδ T-cell Subsets
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Single‐cell suspensions were prepared from inguinal and axillary LNs collected of young and old mice. To enrich γδ T cells, αβ T cells and B cells were depleted from single‐cell suspensions by MACS using a biotinylated antibody against TCRβ with anti‐biotin microbeads and anti‐CD19 microbeads, respectively. Enriched γδ T cells were then stained for FACS sorting as described above. Gating strategy used to identify Vγ1+ γδ1, Vγ4+ γδ1, Vγ4+ γδ17 and Vγ6+ γδ17 T cells is summarised in Appendix Fig S5A and B . γδ T‐cell subsets were sorted with a BD FACS ARIA instrument directly into 3 μl of lysis buffer from SMART‐Seq v4 Ultra Low Input RNA Kit (1 μl of 10× Reaction Buffer and 2 μl of water) accordingly to the instructions of the manufacturer (Clontech). Cells were centrifuged, immediately frozen in liquid nitrogen and stored at −80°C.
7
Apoptosis Quantification by Flow Cytometry
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Cells were seeded in 6-well plates and grown in complete medium, washed with PBS, detached with 0.25% trypsin-0.2% EDTA and centrifuged for 5 min at 1200 rpm. Cells were stained with FITC Annexin V (InVitrogen, Waltham, MA, USA) and Propidium Iodide according to manufacturer’s instructions, and then diluted with Annexin V-binding buffer (InVitrogen, Waltham, MA, USA). Fluorescence intensity was analyzed using BD FACS Aria instrument.
8
CRISPR-Mediated PPP6C Knockout
9
Multiparameter Flow Cytometry Analysis
10
PBMC Isolation and Flow Cytometry
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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood using Ficoll Hypaque. Monoclonal antibody reagents specific for CD3 (UCHT1), CD4 (RPA-T4), CD8 (SK1) and CXCR3 (IC6) were purchased from Becton Dickenson. PBMCs were first incubated with unlabeled human IgG to block nonspecific binding followed by incubation with fluorescent tagged monoclonal reagents. PBMCs were washed and polychromatic flow cytometry performed using a FACSAria instrument (Becton Dickinson, San Jose, CA). Lymphocytes were gated using forward and side scatter and the data analyzed using FlowJo software (Tree Star).
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