Ifn γ pe cy7 4s b3

Cryopreserved PBMCs from IRB consented blood donors were thawed at 37°C, washed twice with complete RPMI media [cRPMI; RPMI 1640 medium (Cellgro) supplemented with 5mM HEPES, 2mM Glutamax (Invitrogen), 50 μg/mL Penicillin (Invitrogen), 50 μg/mL Streptomycin (Invitrogen), 50 M 2-mercaptoethanol (Sigma-Aldrich), and 10% FBS (Atlanta Biologicals)], and plated at 2.5×10⁵ cells per well in cRPMI. Cells were activated with soluble anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL), and treated with IL-12 (1 ng/mL) and/or IL-18 (10 ng/mL) (R&D Systems) for 72h as described previously^{1 (link)}. GolgiStop (4 μL/6 mL culture; BD Biosciences) was added four hours prior to staining for flow cytometric analysis. Cells were first stained for the following surface markers: CD4–Pacific Blue (RPA-T4; eBioscience), TIGIT–APC or –PerCP-eFluor710 (MBSA43; eBioscience), CD226–PE (11A8; BD Biosciences), CD8–APC-Cy7 (SK1; BD Biosciences), and CD25–APC or –AlexaFluor (AF)-488 (BC96; BioLegend). For intracellular staining, cells were fixed, permeabilized, and stained for IFN-γ–PE-Cy7 (4S.B3; BioLegend), Helios–PE, –Pacific Blue, or –AF647 (22F6; BioLegend), and FOXP3–AF488 or –PE (206D; BioLegend) using the FOXP3 Fix/Perm staining kit (BioLegend) according to manufacturer’s protocol. Samples were collected on a BD LSRFortessa and analyzed using FlowJo software.