First, cells were rinsed with FACS buffer and then stained for the cell surface proteins CD4 and CD25 for 30 min at 4 °C. The cells were then fixed and permeabilized by using the fixation/permeabilization kit followed by the Foxp3 antibody incubation for one hour. Cells were subsequently washed and resuspended in FACS buffer for flow cytometry analysis. Before subjecting cells to FACS analysis, all the samples were passed through a 0.40 μm filter and stained with SYTOX Orange Dead Cell Stain (invitrogen). Samples were run on a BD FACSCanto II (Becton and Dickinson) and sorted with a FACSArial cell sorter (Becton and Dickinson) using the Diva software package. Analyses were performed with either Diva or FlowJo_v10 software. Graphs were generated with Prism 8 software (GraphPad Software, Inc.). The antibodies used for staining were anti-human/ mouse CD4 FITC, anti-human CD25 APC, and anti-human Foxp3 PE (eBioscience). Proper controls were also used through the sample preparation process, including a negative control and single-color controls for each antibody.
Facsarial cell sorter
Manufactured by BD
Sourced in United States
The FACSArial cell sorter is a flow cytometry instrument designed for high-performance cell sorting. It is capable of sorting multiple cell populations simultaneously based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Anti human foxp3 pe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti human cd25 apc, by Thermo Fisher Scientific (1 mentions)
Sytox orange dead cell stain, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Staining buffer, by BioLegend (1 mentions)
Diva software package, by BD (1 mentions)
Lsrii cell analyzer, by BD (1 mentions)
Prism 7, by GraphPad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Biolaminin, by BioLamina (1 mentions)
Accutase, by STEMCELL (1 mentions)
Lentivirus, by Genechem (1 mentions)
6 protocols using facsarial cell sorter
1
Regulatory T Cell Phenotyping by Flow Cytometry
2
CD274 Expression Analysis by FACS
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Cells were harvested, washed with PBS and the cell pellet was obtained by brief centrifugation. Cell pellets was then rinsed in 100–200 μL staining buffer (BioLegend, cat no. 420201) and PE-conjugated CD274 antibody was added (BioLegend, cat no. 124308, 1/100). Following 45–60 min incubation on ice, cells were washed with PBS and resuspended in 0.5–1 ml staining buffer. Before subjecting cells to FACS analysis, all the samples were passed through a 0.40 μm filter and additionally stained with DAPI (4′,6-diamidino-2-phenylindole, Invitrogen, cat no. R37606), except for auto-fluorescence control samples. For all samples a ‘full minus one’ (FMO) control was performed50 (link). Samples were run on a LSRII cell analyzer (Becton and Dickinson) and sorted with a FACSArial cell sorter (Becton and Dickinson) using the Diva software package (Becton and Dickinson). Analyses were performed with either Diva or FlowJo_v10 software. Normalized MFI (mean fluorescent intensity) was calculated for all samples relatively to the untreated control group. Graphs were generated with Prism 7 software (GraphPad Software, Inc.).
3
Mouse and Human Cell Immunophenotyping
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Cells from mouse PB, spleen, unfractionated BM or magnetically c-kit enriched BM or human CB cells were stained in PBS (Invitrogen) supplemented with FCS (Invitrogen) together with combinations of antibodies. For full list of antibodies, see supplemental Table 1 . Dead cells were detected by staining with 7-aminoacinomycin D (7-AAD; Sigma-Aldrich. Cells were analyzed using FACSCanto (BD Biosciences). For sorting cells a FACSArial cell sorter (BD Biosciences) was used.
4
Multi-parameter Flow Cytometric Analyses of Leukemic B-cell Progenitors
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For the analyses of p190-BCR-ABL+ (EYFP+) leukemic B-cell progenitors, the PB, BM, and splenocytes of primary and secondary recipient leukemic mice were stained using APC-Cy7–anti–mouse CD45R (clone RA3-6B2, Catalog 552094, BD-Pharmingen), PerCP-Cy5.5–anti–mouse IgM (clone II/41, Catalog 550881, BD-Pharmingen), PE–anti–mouse CD43 (clone S7, Catalog 553271, BD-Pharmingen), and PE-Cy7- anti-mouse CD19 (clone 1D3, Catalog 552854, BD-Pharmingen) antibodies. For the immunophenotypic characterization of p190-BCR-ABL+ leukemic B-cells progenitors in mock/Bmi1 transduced and transplanted mice, the PB, BM and splenocytes were stained for APC-Cy7–anti–mouse CD45R (clone RA3-6B2), PerCP-Cy5.5–anti–mouse IgM (clone II/41), PE–anti–mouse CD43 (clone S7), PE-Cy7- anti-mouse CD19 antibodies, and EGFP+ EYFP+ B-cell progenitors were analyzed. FACS-Canto flow cytometer and the FACSArial cell sorter (both BD Biosciences) were used for analyses and sorting, respectively. For the BM chimera analysis of NSG mice transplanted with Ntg or VAV3 shRNAs (EGFP+) transduced human B-ALL, BM cells were stained for V450-hCD45 (clone HI30, Catalog 560367, BD Horizon), PE-hCD34 (clone 581, Catalog 555822, Biolegend), and APC-Cy7-hCD19 (clone SJ25C1, Catalog 557791, BD-Pharmingen). All antibodies were used at a dilution of 1:100 for 1 × 106 cells.
5
Lentiviral Transduction of GFP into NSCs
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The plasmid Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin was packaged into lentivirus (Genechem Technologies Inc., Shanghai, China) to introduce the green fluorescent protein (GFP) into NSCs. Neurospheres were dissociated into single cells with accutase (Stemcell Technologies Inc., Vancouver, BC, Canada.) and seeded to 6 cm diameter culture dishes at a concentration of 1 × 106 cell/dish. Dishes were coated with biolaminin (10 μg/mL, BioLamina, Sundbyberg, Sweden). After 24 h, NSCs were infected with the lentivirus according to the manufacturer’s instructions. Two days after lentiviral transfection, GFP-positive NSCs were screened in medium containing puromycin (1 μg/mL) and sorted with a FACS Arial cell sorter (BD Biosciences, San Jose, CA, USA). GFP-NSCs were then propagated for at least 5 passages in NSC medium and transplanted into mice hippocampus.
6
Generating Quadruple Knockout MCF7 Cells
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4 μg of pX458-GFP-RIPK1/caspase-8/FADD/cFLIP plasmid was transfected into 1 × 107 MCF7/TO-RIPK3 cells using the Transfection Reagent (FuGENEHD) by following the manufacturer’s instructions. 3 days after the transfection, GFP-positive live cells were sorted into single clones by using a BD FACSArial cell sorter. The single clones were cultured into 96-well plates for another 10–14 days or longer, depending upon the cell growth rate. The anti-RIPK1/caspase-8/FADD/cFLIP immunoblotting was used to screen for the MCF7/TO-RIPK3 (RIPK1-/-/Caspase-8-/-/FADD-/-/cFLIP-/-) clones. Genome type of the knockout cells was determined by DNA sequencing.
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