Propidium iodine

For intracellular staining, pretreated cells were resuspended in complete RPMI and stimulated with 6 μg/mL of plate-bound anti-TCR (anti-CD3 OKT3 clone) for 8, 18, and or 24 h, in the presence or absence of brefeldin A or Monensin (BioLegend) added for the last 6 h. Cells were washed in FACS buffer (PBS, 10% FBS, and 0.05% sodium azide), and then stained for cytokines per manufacturer’s suggestion (Biolegend). All antibodies were purchased from Biolegend. For surface molecule staining, pretreated cells were washed in FACS buffer and stained on ice with the primary and/or secondary antibodies, followed by FACS analysis. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-TCR stimulated cells was less than 1%. For survival assays, pretreated cells were washed in annexin V binding buffer and stained with annexin V and propidium iodine (PI) per manufacturer’s suggestion (BD Biosciences). annexin V and PI double negative cells were considered as viable cells, annexin V positive but PI negative cells as apoptosis undergoing cells, and annexin V and PI double positive cells as dead cells.

Isolated DCs were cultured for 24 h in the presence of Rv0315 or mutant protein (5, 10 or 20 μg/ml). These BMDCs were harvested, washed with phosphate-buffered saline (PBS) and stained with APC-conjugated anti-H-2Kb (MHC class I), FITC-conjugated anti-I-A/I-E (MHC class II), PE-conjugated anti-CD80, and APC-conjugated anti-CD86, along with PE-Cy7-conjugated anti-CD11c (BD Pharmingen) for 30 min at 4 °C. The cells were then washed three times with PBS and resuspended in 500 μl of PBS. Surface markers for BMDC maturation were analyzed by FACSCalibur flow cytometry (BD Biosciences). DCs were stimulated with LPS in medium as a positive control or with medium alone as an untreated control. Cytotoxicity was analyzed by staining for surface-exposed phosphatidylserine with FITC-annexin V in combination with propidium iodine (PI) (BD Biosciences) according to the manufacturer’s instructions.

Cell culture materials including Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Gaithersburg, MD, USA) and Hyclone Laboratories, Inc. (Logan, UT, USA), respectively. Antibodies specifically for p38, phosphorylated p38, caspase 3, propidium iodine (PI), and FITC (fluorescein isothiocyanate-labeled) Annexin V Apoptosis Detection Kit I were obtained from BD Biosciences (San Jose, CA, USA). The Human Apoptosis Array Kit was purchased from R&D Systems (Minneapolis, MN, USA). Additionally, antibodies specifically for ERK1/2, JNK1/2, phosphorylated ERK1/2 and JNK1/2, caspases 8 and 9, and cleaved caspases 3, 8, and 9 were purchased from Cell Signaling Technology (Danvers, MA, USA). Unless otherwise specified, all chemicals used in this study, including streptomycin, were purchased from Sigma-Aldrich (St. Louis, MO, USA).