Fix perm staining kit

Cell surface stains were performed by washing cells twice with FACS buffer, 1% FBS in 1× PBS, followed by incubation with human Cohn's fraction IgG (Sigma-Aldrich, G4386) for 10 minutes to reduce non-specific binding. Samples were then washed with FACS buffer twice before incubation in the appropriate mixture of antibodies for 30 min at 4°C. After incubation, the cells were washed three times and resuspended in FACS buffer before samples were acquired on a C6 Accuri flow cytometer (BD Biosciences, Ann Arbor, MI). Expression of MICA/B on cell lines was quantified using PE-conjugated anti-MICA/MICB antibody clone 6D4 (Biolegend, 320906). T cell subsets were identified using anti-CD3 clone OKT3 conjugated with PerCP 710 (eBiosciences, 46-0037-42), PE conjugated anti-CD45RO clone UCHL1 (Biolegend, 304206), and FITC-conjugated anti-CCR7 clone 150503 (R&D Systems, FAB197F). When intracellular staining was performed, cell surface stains were completed before cells were fixed and permeabilized using a Fix/Perm staining kit (eBioscience, 00-5523-00) followed by intracellular staining for IFNγ with APC anti-human IFNγ clone B27, (Biolegend, 506510) or isotype control APC msIgG1 clone MOPC-21 (Biolegend, 400120) per the manufacturer's instructions. All data acquisition and analysis was done using the Accuri flow cytometry software (BD Biosciences, Ann Arbor, MI).

Lymph node cells were analyzed using an LSR II or Fortessa instrument (BD) and FlowJo software (Tree Star, Inc.). Typically, RBC-depleted spleen cell suspensions were prepared and washed with isotonic buffer (Yenson and Baumgarth, 2014 (link)). Cell suspensions were stained with antibodies listed in Key Resources Table. For sorting, donor OT-II cells were sorted on the FACSAria (BD). Detection of Irf4, Bcl6, and TBET was performed by fixing and permeabilizing cells with Fix/Perm staining kit (eBioscience). CD45.1⁻B220⁻MHCII⁻CD45.2⁺CD4⁺ donor cells were measured for IFNγ and IL-2 cytokine expression after stimulation with PMA/Ionomycin for 6 hours in the presence of monensin and brefeldinA. Statistical analyses using non-parametric unpaired t-tests were performed with Prism.