Antibody staining of single-cell suspensions was performed as previously described(21 (link)). Foxp3 intracellular kit (eBioscience) was used following manufacturer’s instructions(19 (link)). For intracellular staining, PE anti-Granzyme B or isotype control (Invitrogen, Grand Island, NY) was used. Stained cells were analyzed with an LSR-Fortessa cytometer (Becton Dickinson, San Jose, CA) and FlowJo software (TreeStar) was used for data analysis.
Foxp3 intracellular kit
Manufactured by Thermo Fisher Scientific
The Foxp3 intracellular kit is a laboratory product designed for the detection and analysis of the Foxp3 transcription factor. The kit provides a method for intracellular staining and flow cytometric analysis of Foxp3 expression in cells.
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Lab products found in correlation
Ghost dye violet 510, by Cytek Biosciences (2 mentions)
Lsrfortessa, by BD (2 mentions)
Accuri c6, by BD (2 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Anti granzyme b pe, by Thermo Fisher Scientific (1 mentions)
Isotype control, by Thermo Fisher Scientific (1 mentions)
Foxp3 fixation buffer, by Thermo Fisher Scientific (1 mentions)
Calibrite beads, by BD (1 mentions)
Rat serum, by Thermo Fisher Scientific (1 mentions)
Icam1 pe, by Thermo Fisher Scientific (1 mentions)
Pd l1 pe, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Cd105 apc, by Thermo Fisher Scientific (1 mentions)
5 protocols using foxp3 intracellular kit
1
Single-cell Foxp3 and Granzyme B staining
2
Multiparametric Flow Cytometry Analysis
3
Multiparameter Flow Cytometry Analysis
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Fluorescence antibody-stained single-cell suspensions were analyzed using LSRFortessa or LSRII flow cytometers (BD Biosciences). For live cell analysis, dead cells were excluded by adding propidium iodide before running the samples on flow cytometers. For fixed cell staining and analysis, cells were stained with Ghost Dye Violet 510 (Tonbo) for exclusion of dead cells, followed by surface staining and fixation with Foxp3 fixation buffer (eBioscience). Afterwards, cells were permeabilized using reagents from the Foxp3 intracellular kit according to the manufacturer's instructions (eBioscience). Excess reagents were removed by extensive washing in FACS buffer (0.5% BSA, 0.1% sodium azide in HBSS) before analysis.
4
Immunosuppressive Potential of MSCs on PBMC Proliferation
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Human PBMCs were isolated from buffy packs (Irish Blood Transfusion Service), by Ficoll density gradient centrifugation. 5 × 104 Carboxyfluorescein succinimidyl ester (CFSE) labelled PBMC were co-cultured (Fisher, Ballycoolin, Ireland) with MSC (1 × 104/well) (1:5). In the presence of CD3/CD28 Dynabeads® beads (Gibco) (1 × 104/well). After 4 days, PBMC were harvested and the level of proliferation by CD3+ cells was analysed by flow cytometry (Accuri C6, BD Biosciences) and enumerated using counting beads (3 × 105/ml) (Calibrite™ Beads, BD Biosciences). To examine MSC production of IDO following co-culture with activated PBMC, MSC were labelled with cell proliferation dye eFluor® 670. For analysis of intracellular IDO, cells were incubated with 1X Brefeldin A (eBioscience) for the last 4 h of the co-culture on the third day of the PBMC suppressor assay followed by preparation using the intracellular FoxP3 kit (eBioscience) as per manufacturer’s instructions and incubation with IDO PE antibody (eBioscience) for 45 min. Cells were then washed in FACs buffer and acquired using the Accuri C6 gating on the cell proliferation dye eFluor® 670 labelled human MSC.
5
Multiparametric Phenotyping of MAPCs
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MAPCs were cultured, transferred to v bottom 96‐well plates and washed twice in flow cytometry staining buffer (PBS containing 2% FBS). Cell pellets were dissociated briefly by vortexing and antibodies for CD105 APC, ICAM1 PE, or PDL1 PE (eBioscience, Part of ThermoFisher Scientific) were added. Cells were incubated with surface antibodies for 30 minutes at 4°C, and then washed in flow cytometry staining buffer. Cells were then ready to acquire by flow cytometry on an Accuri C6. For intracellular staining, cells were incubated with 1X Brefeldin A (eBioscience, Part of ThermoFisher Scientific) for 4 hours before harvest. The intracellular FoxP3 kit was used per manufacturer's instructions (eBioscience, Part of ThermoFisher Scientific) to prepare cells for intracellular staining. Cells were blocked with 2% rat serum (eBioscience, Part of ThermoFisher Scientific) for 15 minutes to prevent nonspecific staining, and either IDO or COX‐2 antibodies (BD Biosciences, Berkshire, UK) were then added for 45 minutes. Cells were then washed in flow cytometry staining buffer and acquired using the Accuri C6 (BD Biosciences).
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