Ionomycin cell activation cocktail

Cells were placed in 250 μL RPMI supplemented with 10% FBS, 2mM Glutamine (Gibco), 2mM Pyruvate (BioWhittaker), 50 μg/mL Pen/Strep (BioWhittaker), and 0.55 mM 2-ME (Gibco). 1X protein transport inhibitor (Fisher Healthcare) and 1:1000 phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Cell Activation Cocktail, Biolegend) were added, and samples were placed in an incubator at 37°C for four hours. Samples were spun down and resuspended in FACS buffer to block for 15 minutes. Samples were then stained for surface markers for 30 minutes at 4°C before 100 μL of fixative (Life Technologies) was added to each sample for 30 minutes at room temperature. Samples were then washed once with FACS buffer and once with 1X perm buffer (Biolegend) and spun down at 1500 rpm for 5 minutes. Samples were then resuspended in 100 μL 1X perm buffer and intracellular antibodies and were placed at 4°C overnight. The following day, samples were washed twice with FACS buffer and acquired on the CyTek Northern Lights Spectral Flow Cytometer (CyTek Biosciences).