T-cell proliferation assay

Lymphocytes were obtained from spleens of WT mice intravaginally infected with Chlamydia using a 40 μm filter and syringe plunger and suspended in PBS solution. The CD4⁺ T cells were then purified using the MACS mouse Pan T Cell Isolation Kit (mouse) (Miltenyi Biotec, Inc, San Diego, CA). To assess the antigen-presenting function of WT and ENO1 knockdown DCs, 1 × 10⁵ γ-irradiated DCs were co-cultured with 2 × 10⁵ purified T cells in the presence or absence of UV-inactivated C. muriduram (MOI of 5) in 96-well tissue culture plates for 5 days. The amounts of IL-1β, IL-1α IL-5, IL-9, IL-10, IL-13, IL-17A, and RANTES in the culture supernatants were measured using the Bio-Plex Pro Mouse Cytokine 23-Plex multiplex array according to the manufacturer’s guidelines (Bio-Rad) using a Luminex machine. The T cell proliferation was detected using a spectrophotometer set at 450 nm following the XTT Cell Viability Kit protocol (Cell Signaling Technology, Danvers, MA). The concentration of cytokine in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. The mean and SD of all replicate cultures were calculated. The experiment was repeated two times.