Percp cy5.5 conjugated cd4

Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). DMEM was obtained from HyClone Co. (Logan, UT, USA). The enzyme-linked immunosorbent assay (ELISA) kits for immunoglobulin A (IgA), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were purchased from R&D Systems (Minneapolis, MN, USA). The antibodies, including APC-conjugated CD3, PerCP-Cy5.5-conjugated CD4 and PE-conjugated CD19, were obtained from Biolegend (San Diego, CA, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and are all analytical grade.

Freshly isolated splenocytes (1 × 10^E6) were seeded in 96-well plates and stimulated with 5 μg/mL Ag85B antigen or 1 μ/mL ESAT-6 antigen. As positive control, the cells were stimulated with 1 μg/mL α-CD3 (BioLegends). Antigen-specific T cell proliferation was analyzed after 6 days of incubation with antigens. The cells were blocked, as described above, and subsequently stained with PerCP/Cy5.5-conjugated CD4, Brilliant Violet 510™-conjugated CD8a, FITC-conjugated CD44, PE-conjugated CD62L and Brilliant Violet 421™- conjugated CD90.2 antibodies (all from BioLegends), all diluted 1:100. After staining, the cells were fixed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and permeabilized using eBioscience™ Permeabilization Buffer, according to the manufacturer's instructions. Subsequently, cells were intracellularly stained with 1:50 diluted APC-conjugated Ki-67 antibody (BioLegends) and analyzed by flow cytometry.