Cxp analysis software

LNCs from PLP- immunized HIIT and SED mice were analyzed for surface integrin expression following their activation in vitro for 72 h in culture with PLP peptide, using flow cytometry (Figs. 1B and 4I–K,M–O; n = 8/group). The following antibodies were used: FITC-labeled anti-CD4 (anti- mouse CD4, 100,405, BioLegend) and PE-labeled anti- VLA-4 (anti- mouse CD49d, 103,607, Biolegend), APC-labeled anti- LFA-1 (anti- human CD11a/CD18, 363,409, BioLegend), FITC-labeled anti-F4/80 (anti- mouse F4/80, 123,107, BioLegend), PE-labeled anti-B220 (anti- CD45R, 45-0452-82, eBiosciense). The fraction of VLA-4+ or LFA-1+ cells out of total LNCs was calculated. Double staining with anti-VLA-4 and anti-CD4 or anti-F4/80 was used to identify the fraction of VLA-4+ T cells or macrophages, respectively. Double staining with anti-LFA-1 and anti-CD4 or anti-B220 was used to identify the fraction of LFA-1+ T cells or B cells, respectively. In all flow cytometry experiments, cells were pre-coated with anti–mouse CD16/CD32 (BD Pharmingen), as an Fc blocker, to block non-specific binding. In early experiments antibodies were tested for their specificity with an isotype control. All samples were analyzed in a Cytomics FC 500 apparatus (Beckman Coulter, Life Science) using the CXP analysis software (ver. 2.1; Informer Technologies, Inc).