LPS and PMA/ionomycin stimulation

After thawing, cells were counted and resuspended in OpTmizer medium (cat# A1048501; ThermoFisher Scientific, Waltham, MA) at 2 × 10⁶ cells/ml, and 100 µl aliquots were plated in a 96-well round-bottom plate. After 1–3 h rest at 37°C, 100 µl of OpTmizer with 2× stimulant concentration was added, and cells were returned to 37°C. The final concentration of stimulants was 10 ng/ml LPS (cat# tlrl-3pelps; Invivogen, San Diego, CA), 10 ng/ml PMA, and 0.5 µg/ml ionomycin (cat# P8139 and cat# I0634, respectively; both from Sigma-Aldrich, St. Louis, MO). After 24 h, cells were briefly mixed and then spun down at 974 × g for 5 min, and the supernatant was frozen at −80°C.