Legendplex mouse 8 plex th1 th2 panel

Cytokines secreted into the culture supernatant were quantified using a LEGENDplex mouse 8-plex Th1/Th2 panel (BioLegend) following the manufacturer’s protocol. Samples were acquired using a BD Accuri C6 flow cytometer (BD Biosciences), and data were analyzed using LEGENDplex Data Analysis software (BioLegend v8.0). The concentration of cytokines due to antigen stimulation was calculated by subtracting the concentration of cytokines in unstimulated samples from the concentration in stimulated samples.

RAW264.7 macrophages seeded into 6-well plates (Costar, Cambridge, MA, USA) at the density of 7 × 10⁵ cells/well were infected with MSmeg strains at the MOI 10:1 for 3 h, washed five times with PBS, and cultured for the indicated times. RNA extraction and purification from infected macrophages was performed with TRIzol (Evrogen) using the standard phenol-chloroform method. cDNA synthesis and qRT-PCR were performed as described in Section 2.5. Relative mRNA levels were calculated after normalization to β-actin. PCR primers are listed in Supplementary Table S1.
To evaluate IL-6 secretion in culture supernatants of infected macrophages, samples were analyzed with the LEGENDplex mouse 8-plex Th1/Th2 panel (BioLegend, San Diego, CA, USA) following the manufacturer’s protocol, and acquired using BD FACSCalibur flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA, USA). The data were processed using WinMDI2.8 software (The Scripps Institute, West Lafayette, IN, USA).