This protocol outlines the steps used for Intracellular cytokine staining.In brief: Cells were treated with protein transport inhibitor containing Brefeldin A (GolgiPlug; cat: 555029, BD) and plated at 1e6 cells per well in a 96-well flat bottom plate. Cells were stimulated for 5 hours at 37\u00b0C with either T cell media, Phorbol 12-Myristate 13-Acetate (Sigma, cat: P8139) (used at 20ng\/mL) and Ionomycin (Sigma, cat: I0634)(used at 1mg\/mL), or 1mg\/mL of peptide (as indicated). Msln406-414 (GQKMNAQAI), OTI (SIINFEKL), LCMVgp33-41 (KAVYNFATM), MSLN20-28 (SLLFLLFSL), and MSLN530-538 (VLPLTVAEV) peptides were ordered from ELIM peptide (>80% purity). The BD Fix\/Perm kit (cat: 554714) was used for intracellular staining. Cells were fixed in 0.5% paraformaldehyde until data acquisition.