Spleen cells from OT-I, in which the TCR of CD8+ cells are restricted to OVA were isolated and labeled with 10 μM CFSE. Half a million of these cells were transferred intravenously into recipient mice 1 day before intranasal delivery of apoptotic cells and sOVA. Mice were sacrificed 3 days after intranasal deliveries for analysis of T cell proliferation (CFSE dye dilution) in the LLN. For intracellular cytokine staining, LLNs were isolated 3 days after intranasal deliveries of apoptotic cells or sOVA ± Poly I:C and digested in Collagenase D for 30 min, then pressed through a 100 μm nylon filter to obtain single cell suspensions of antigen presenting cells and proliferating T cells. Isolated cells were cultured 5h in RPMI 10% FCS containing 10 μM OVA peptide (257-264) and Brefeldin A 10 μg/ml. Following surface staining, BD Foxp3 intracellular staining kit was used. Cells were stained with APC-conjugated to IFNγ, PerCPCy5.5-TNFα, PECy7-granzymeB or isotype controls (BD Pharmingen).
Foxp3 intracellular staining kit
Manufactured by BD
The BD Foxp3 intracellular staining kit is a laboratory tool used to detect and quantify regulatory T cells (Tregs) by flow cytometry. It provides a method for the intracellular staining and detection of the Foxp3 transcription factor, a key marker for identifying Tregs. The kit includes the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
3 protocols using foxp3 intracellular staining kit
1
Tracking CD8+ T cell Proliferation
2
Tracking CD8+ T cell Proliferation
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Spleen cells from OT-I, in which the TCR of CD8+ cells are restricted to OVA were isolated and labeled with 10 μM CFSE. Half a million of these cells were transferred intravenously into recipient mice 1 day before intranasal delivery of apoptotic cells and sOVA. Mice were sacrificed 3 days after intranasal deliveries for analysis of T cell proliferation (CFSE dye dilution) in the LLN. For intracellular cytokine staining, LLNs were isolated 3 days after intranasal deliveries of apoptotic cells or sOVA ± Poly I:C and digested in Collagenase D for 30 min, then pressed through a 100 μm nylon filter to obtain single cell suspensions of antigen presenting cells and proliferating T cells. Isolated cells were cultured 5h in RPMI 10% FCS containing 10 μM OVA peptide (257-264) and Brefeldin A 10 μg/ml. Following surface staining, BD Foxp3 intracellular staining kit was used. Cells were stained with APC-conjugated to IFNγ, PerCPCy5.5-TNFα, PECy7-granzymeB or isotype controls (BD Pharmingen).
3
In Vivo T Cell Proliferation Assay
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Spleen cells from OTI or OTII GFP mice, in which the TCR of CD8+ or CD4+ T cells are restricted to an OVA peptide, were labeled with 10 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 mins. Half a million of these cells were transferred intravenously into recipient mice 1 day before intranasal delivery of 2 μg soluble OVA and 20 μg Poly I:C. Mice were sacrificed 3 days after intranasal deliveries for analysis of T cell proliferation (CFSE dye dilution) in the LLN. For OTI: Isolated cells were cultured 5 hours in RPMI 10% FCS containing 10 μM OVA peptide (257-264) and Brefeldin A 10 μg/ml. Following surface staining, BD Foxp3 intracellular staining kit was used. Cells were stained with APC-conjugated to IFNγ, PerCP-Cy5.5-TNFα, or isotype controls (BD Pharmingen). For OTII: Isolated cells were cultured 5 hours in RPMI 10% FCS containing 10 μM OVA peptide (323-339) and Brefeldin A 10 μg/ml. Following stimulation, cells were stained with PE-conjugated to CD4, APC-conjugated to Vβ51, PE-Cy7-conjugated to Vα2, APC-Cy7-conjugated to CD44, BUV805-conjugated to CD8 (BD Pharmingen, eBioscience or Biolegend).
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