Fitc conjugated control sirna

Polarized CD27⁻ γδ T cells were cultured in RPMI 1640 medium with 20% fetal bovine serum (FBS), and fluorescein isothiocyanate (FITC)–conjugated control siRNA (Santa Cruz Biotechnology, sc-36869) and IDH2 siRNA (Thermo Fisher Scientific, AM16708) were transfected with Lipofectamine 2000 (11668-027, Invitrogen). For the cytokine stimulation, cells were cultured for additional 24 hours with IL-1β (10 ng/ml) and IL-23 (10 ng/ml). The Golgi plug was added in the final 4 hours. For the PMA stimulation, cells were cultured for additional 24 hours and stimulated with PMA and ionomycin in the presence of Golgiplug (BioLegend) for 4 hours. Intracellular cytokine staining of IL-17 was performed. For lentivirus infection, polarized CD27⁺ γδ T cells were cultured in complete RPMI 1640 medium with polybrene (8 μg/ml). Cells were infected with GFP-tagged blank lentivirus or IDH2 lentivirus (Applied Biological Materials Inc.) by centrifugation at the speed of 2300 rpm for 90 min at 30°C. Cells were then cultured in RPMI 1640 medium containing 20% FBS, IL-2 (10 ng/ml), and IL-7 (10 ng/ml). On the second day, GFP⁺ γδ T cells were sorted and cultured for an additional 48 hours, and intracellular IFN-γ was examined.