System 2 software

Splenic single-cell suspensions prepared from all mice prior to the challenge were used to analyze T lymphocytes using flow cytometry, as previously described [22 (link)]. Briefly, 10⁵ viable cells counted using a hemocytometer and 0.04% trypan blue dye (Bio-Rad) were suspended in 100 μL of 2% FBS in PBS solution and incubated with 0.45 μL of FITC anti-mouse CD3e, 0.25 μL of APC-eFluor780 anti-mouse CD4 or 0.25 μL of Percp-cy5.5 anti-mouse CD8a (eBioscience, San Diego, California, USA) at 4 °C for 30 min in the dark. After two washes with 2 mL of PBS, the samples were re-suspended in 300 μL of FACScan buffer (0.1% NaN₃, 1% BSA) and 250 μL of 2% PFA solution for optical measurement. The data from three independent experiments were obtained using the FACScan flow cytometer and SYSTEM II software (BD Bio-sciences, San Jose, California, USA).