Mature CD8SP or CD4SP cells from ATOs were isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naïve T cells and CD4ISP precursors). Purified T cell populations were plated in 96-well U-bottom plates in 200 μl AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) were added to each well and incubated for 6h. Cells were stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).
Cd8 or cd4 isolation kits
Manufactured by Miltenyi Biotec
The CD8+ or CD4+ Isolation Kits are laboratory equipment designed for the isolation and separation of CD8+ or CD4+ T cells from a mixed cell population. The kits utilize magnetic bead-based cell separation technology to efficiently isolate the target cell subsets.
Automatically generated - may contain errors
Lab products found in correlation
Control protein transport inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Intracellular staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Aim 5, by Thermo Fisher Scientific (2 mentions)
Pma ionomycin protein transport inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Uv455 fixable viability dye, by Thermo Fisher Scientific (2 mentions)
Human ab serum, by Gemini Bio (2 mentions)
2 protocols using cd8 or cd4 isolation kits
1
Intracellular Cytokine Profiling of Mature T Cells
2
Intracellular Cytokine Profiling of Mature T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Mature CD8SP or CD4SP cells from ATOs were isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naïve T cells and CD4ISP precursors). Purified T cell populations were plated in 96-well U-bottom plates in 200 μl AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) were added to each well and incubated for 6h. Cells were stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).
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