Human il 12

PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics (at 37 °C and 5% CO₂ in humidified incubator) and pulsed with a range of stimulants. For cytokine stimulation: 100 U/mL human IL-2 (Abcam), 200 ng/mL human IL-12 (Miltenyi Biotec) and 200 ng/mL human IL-18 (BioLegend) were pulsed in two doses and cultured 72 h apart, and brefeldin A (BFA) (Invitrogen) added after second dose, before culturing for 18 h. For bacterial stimulation: E. coli at multiplicity of infection (MOI) 50 was added to PBMCs, cultured for 1 h prior to adding 50 μg/mL gentamicin (Sigma) and BFA and cultured for 18 h. For antigen stimulation: 10 ng/mL of HMBPP (Cayman Chemicals) was added and cultured for 4 h before adding BFA and cultured for 18 h. For phytohemagglutinin (PHA) stimulation: PHA-L (eBioscience) was added at 1× working concentration and cultured for 1 h prior to addition of BFA and cultured for 18 h. Following stimulation, cells were collected and stained with viability dye for 10 min followed by surface antibodies for 20 min on ice. Cells were fixed and permeabilized using the eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Invitrogen). Intracellular antibodies were then incubated for 45 min at room temperature diluted in permeabilization buffer.