Splenocytes were prepared and counted from each mouse in different groups as described previously (32 (link)). Subcomponent-specific cytokines Th1 (TNF-α and IFN-γ), Th2 (IL-4), and Th17 (IL-17A) were detected using cytometric bead array (CBA). 5 × 106 cells were plated into each well of a 24-well plate and incubated with RPMI1640 medium (negative control), or RPMI1640 medium with specific proteins including Rv0577, Rv2875, Rv3044, or Rv2073c (each 10 µg/mL), respectively. The supernatant was collected 24 h later and Cytometric Bead Array kit (BD Biosciences, NJ, USA) was performed according to the manufacturer’s instructions.
Alternatively, 5 × 106 cells were incubated with CMFO protein (10 µg/mL) at 37°C and 5% CO2. RPMI1640 medium was used as a negative control and PPD (10 mg/mL, Statens Serum Institut, Denmark) was set as a positive control. After an incubation of 72 h, the supernatant was collected and a commercial mouse IFN-γ ELISA kit (Multi Sciences, Hangzhou, China) were used to detect the concentration of IFN-γ (20 (link)–22 (link)). The results are expressed as mean ± SD (pg/mL) for each group (n = 6).
Alternatively, 5 × 106 cells were incubated with CMFO protein (10 µg/mL) at 37°C and 5% CO2. RPMI1640 medium was used as a negative control and PPD (10 mg/mL, Statens Serum Institut, Denmark) was set as a positive control. After an incubation of 72 h, the supernatant was collected and a commercial mouse IFN-γ ELISA kit (Multi Sciences, Hangzhou, China) were used to detect the concentration of IFN-γ (20 (link)–22 (link)). The results are expressed as mean ± SD (pg/mL) for each group (n = 6).
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