Pma iono

The human DLBCL cell lines BJAB, U2932, OCI-Ly3 were obtained from DSMZ, SUDHL-4, and SUDHL-6 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium supplemented with 20% FBS and 100 U/ml penicillin/streptomycin (Gibco). OCI-Ly10 was purchased from Cobioer Biosciences Co., LTD (Nanjing, China) and cultured in IMDM with 20% FBS, 100 U/ml penicillin/streptomycin (Gibco) and 50 μM β-mercaptoethanol (Sigma-Aldrich). All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO₂.
z-VRPR-fmk (Enzo Life Sciences) was dissolved in ddH₂O at a concentration of 50 μM throughout all experiments. MI-2, BPTES, QNZ, CPI-613 (Selleck), and PMA/Iono (Sigma-Aldrich) were reconstituted in DMSO (final DMSO concentration 0.1%) and their final concentrations were 1 μM, 2 μM, 5 μM, 100 μM, and 50/500 ng/ml, respectively.

Spleen and BM cells were incubated at 37°C in 5% CO₂ in round-bottom 96-well plates (2×10⁶ cells/well) for 5 hours with a pool of gag protein-derived peptides (gag peptide pool, 15mers overlapping by 11 amino acids) at final concentration of 2 µg/ml for each peptide. Dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), the peptide pool diluent, was used as negative control and phorbol myristate acetate/ionomycin (PMA/Iono, Sigma-Aldrich) at final concentration of 20 ng/ml and 1 µg/ml respectively as positive controls. All incubations were performed in the presence of Golgi plug (BD Biosciences). After stimulation, cells were collected and incubated with Fc block, stained with Live/Dead Fixable Violet Dye (Invitrogen, Thermo Fisher Scientific) for viability, and with the following mAbs against surface markers: anti-CD3ε APC, clone 145-2C11; anti-CD8α PerCP, clone 53-6.7; anti-CD4 PE, clone H129.19 (all from BD Biosciences). Intracellular staining was performed after treatment with Cytofix/Cytoperm and in the presence of PermWash (BD Biosciences) using anti-mouse IFN-γ FITC, clone XMG1.2 (BD Biosciences).