Cytokine mouse 20 plex panel

Animals underwent a submandibular bleed and systemic cytokines were measured by single-plex ELISA (eBioscience, CA, USA or Biolegend, CA, USA) according to the manufacturer’s instructions or by Cytokine Mouse 20-Plex Panel (Life Technologies, Carlsbad, CA) on Luminex Bead Array technology.

Splenocytes were isolated from immunized and non-immunized C57BL/6 mice (6 mice/group), 6 weeks post the last immunization, as previously described.^{30 (link)} Briefly, spleens were minced, and red blood cells lysed with ACK lysing buffer (Cambrex Bio Sciences, East Rutherford, NJ, USA). Splenocytes were cultured at 2×10⁶ cells/mL in Dulbecco’s Modified Eagles Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 2.2 g/L sodium bicarbonate, 0.05 g/L of HEPES (Sigma, St. Louis, MO, USA), 0.05 g/L L-arginine (Sigma), 100 μg/mL penicillin G (Sigma), 50 μg/ mL gentamycin sulfate (Sigma) 0.005% 2-mercaptoethanol (2-Me, Gibco, Carlsbad, CA, USA), and 10% fetal bovine serum (FBS, Sigma). Assay controls were performed to assess potential for cellular activation, including stimulation with ConA (2 μg/ mL), LPS (200 ng/mL). Splenocytes that did not response to ConA and/or LPS were discarded from the analysis. To access recall to BCG antigens, splenocytes were stimulated with heat-killed BCG at 10:1 (organisms:cells) ratio for 72 h. Supernatants were collected and stored at −20°C for later analysis using the Cytokine Mouse 20-Plex Panel (Life Technologies, Grand Island, NY, USA). The multi-bead assay was analyzed using the Luminex® 100/200^TM System according to manufacturer’s instructions.