Pma ionomycin protein transport inhibitor cocktail

Mature CD8SP cells from PSC-ATOs were isolated by magnetic negative selection using the CD8+ T Cell Isolation Kit
(Miltenyi, Cat. 130–096-495) and sorted by FACS to further deplete CD45RO+ cells (containing immature CD8SP T cells and
DN/4ISP T cell 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 washed and stained for CD3, CD4, and CD8 (Biolegend, San Diego,
CA) and Zombie UV™ Fixable Viability dye (Biolegend, 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, and IL-2 (Biolegend, San Diego, CA).

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).

Mature CD8SP and mature CD4SP cells from M-ATOs were isolated by
magnetic negative selection using the CD8⁺ T Cell Isolation Kit
(Miltenyi Biotech, Cat# 130–104-075) and the
CD4⁺ T cell Isolation Kit (Miltenyi Biotech, Cat#
130–104-454) respectively and sorted by FACS or magnetic
selection (Miltenyi Biotech, Cat# 130–091-7558) to
further isolate CD8SP CD62L⁺ cells and CD4SP CD62L⁺cells. Purified T cell populations were plated in 96-well U-bottom plates in
200 μl RPMI 1640 (CellGro, Cat# 10–040-CV) with
5% fetal calf serum (Hyclone, Cat# SH30070.03) and 0.05mM beta
mercaptoethanol (bME) (Sigma-Aldrich, Cat# M7522).
PMA/ionomycin/protein transport inhibitor cocktail or control protein
transport inhibitor cocktail (eBioscience, San Diego, CA, Cat#
00–4975-03) were added to each well and incubated for 6h.
Cells were washed and stained for CD3, CD4, and CD8 (Biolegend, San
Diego, CA) prior to fixation and permeabilization with an
intracellular staining buffer kit (eBioscience, San Diego, CA, Cat#
88–8824-00) and intracellular staining with antibodies
against IFNγ, TNFα, and IL-2 (Biolegend, San Diego,
CA).