Ionomycin inh ion

Splenocytes were rested in complete IMDM (cIMDM) (Sigma) containing 10% FBS, 10 U/ml Penicillin and 10 μg/ml Streptomycin, 40 μM β-Mercaptoethanol and 10 mM HEPES. Following centrifugation, splenocytes were re-suspended and 8.5–10 × 10⁵ cells were seeded in 96-well flat-bottom plates. Following overnight culture at 37°C, splenocytes were stimulated with an agonist array; either 10 μg/200 μl lipopolysaccharide (LPS) from Escherichia Coli 011:B4 (tlrl-eblps, Invivogen), 10 μg/200 μl Concanavalin A (ConA) (C0412, Sigma), 1 μg/200 μl Phorbol myristaste acetate (PMA) (tlrl-pma, Invivogen) mixed with 10 μg/200 μl Ionomycin (inh-ion, Invivogen) or transfected with 1 μg/200 μl high molecular weight polyinosinic:polycytidylic acid (poly I:C) (tlrl-pic, Invivogen) using 3 μl Lipofectamine 3000 (L3000001, LifeTechnologies). After 6 h, RNA was extracted with use of TRIzol Reagent (10296028, Invitrogen) according to manufacturer's instructions. The RNA pellet was visualized by the addition of 1 μl of glycogen (10901393001, Roche) and re-suspended in 40 μl RNase free water. RNA yields ranged from 20 to 25 ng/μl as measured with high sensitivity Qubit kits (Q32852, ThermoFisher).