Cells were plated in 96-well round-bottomed plates and pulsed with peptide antigen for 6 hr at 37°C in T-cell media (RPMI 1640, 10% FBS, 50 μM β-mecaptoethanol, 100 U/mL Penn/Strep, 1x Gibco® MEM Non-Essential Amino Acids Solution (Life Technologies), 1 mM Sodium pyruvate, 1 mM HEPES), followed by the addition of brefeldin A for 4 hours. Cells were stained with anti-CD4-APC and then fixed using Cytofix (BD biosciences) according to the manufacturer’s instructions. Cells were then washed and permeabilized. Intracellular staining for anti-IFN-γ-PE was performed according to the manufacturer’s instructions. Foxp3 staining was performed according to the manufacturer’s instructions (ebioscience) for fixation and permeabilization.
Foxp3 staining
Manufactured by Thermo Fisher Scientific
The Foxp3 staining is a lab equipment product designed for the detection and analysis of Foxp3, a transcription factor that is widely used as a marker for regulatory T cells. The product facilitates the identification and quantification of Foxp3-expressing cells in various biological samples, which is important for research in areas such as immunology and cell biology.
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Lab products found in correlation
Cytofix, by BD (1 mentions)
Transcription staining buffer set, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Cxcr3 173, by BioLegend (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions)
Facscanto system, by BD (1 mentions)
3 protocols using foxp3 staining
1
Intracellular Cytokine Staining Assay
2
Comprehensive Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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FITC-, PE-, PE-CY7, APC-, APC-CY7 or Percp-labeled antibodies to CD3 (145-2C11), CD45 (30-F11), CD4 (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), B220 (RA36B2), CD25 (PC61), NK1.1 (PK136), PD-L1 (MIH5), FoxP3 (NRRF-30), IFN-γ (XMG1.2), TNF-α (MAb11), CD49 (DX5), and CXCR3 (CXCR3-173) and isotype-matched control antibodies were purchased from Biolegend (antibodies to CD3, CD4, CD8, CD45,CD11b, CD11c, B220, CD25, PD-L1, FoxP3, IFN-γ, TNF-α, CD49, CXCR3, and control antibodies) or BD Biosciences (antibodies to NK1.1 and control antibodies). For identification of cellular phenotypes, disassociated cells from tumors or spleens were suspended in PBS containing 1% bovine serum albumin and incubated with the antibodies on ice for 30 min. Cells were fixed in 1% paraformaldehyde in PBS after washing. For intracellular cytokine staining, cells were stimulated in culture medium for 4 h in the presence of Leukocyte Activation Cocktail, with BD GolgiStop (1: 500; BD Biosciences). Viable cells were then fixed and permeabilized with transcription staining buffer set (eBioscience) and stained with respective antibodies to cytokines. FoxP3 staining was performed according to manufacturer’s protocol (eBioscience). Stained cells were analyzed on a FACSCalibur or FACS Canton flow cytometer, and data were analyzed using the flowjo software.
3
Flow Cytometric Analysis of T-Cell Subsets
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