Percp conjugated anti mouse cd4

The following antibodies were purchased from eBioscience: Pacific Blue conjugated anti-mouse CD4 (clone: RM4-5), FITC conjugated anti-mouse CD45.1 (clone: A20), biotin conjugated anti-mouse CD3 (clone: 17A2), FITC conjugated anti-mouse CD3 (clone: 145-2C11), APC conjugated anti-mouse CD317 (BST2, PDCA1) (clone: eBio927), Pacific Blue conjugated anti-mouse CD11c (clone: N418), FITC conjugated anti-mouse MHCII (I-A) (clone: NIMR-4), biotin conjugated CD317 (BST2, PDCA1) (clone: eBio927), PerCP-Cy5.5 conjugated anti-mouse Ly-6G (Gr-1) (clone: RB6-8C5), streptavidin conjugated PerCP-Cy5.5. The following antibodies were from BD Pharmingen: PE-Cy7 conjugated anti-mouse CD45 (clone: 30-F11), PerCP conjugated anti-mouse CD4 (clone: RM4-5), PE conjugated anti-mouse CD19 (clone: 1D3), APC-Cy7 conjugated anti-mouse CD11b (clone: M1/70), Biotin conjugated anti-mouse Ly-6C (clone: AL-21), PerCP-Cy5.5 conjugated anti-mouse CD45R (B220) (clone: RA3-6B2), PE conjugated anti-mouse Ly-6G (clone: 1A8), PerCP-Cy5.5 conjugated Ly-6C (clone: AL-21), streptavidin conjugated V500. Diphtheria toxin (DT, isolated from Corynebacterium diphtheriae) was purchased from Sigma-Aldrich (St Louis, MO). Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences, California, USA) was obtained from BD^TM Cytometric Bead Array (CBA).

T cell activation was assessed by staining the cells for CD4, CD8, and CD62L markers. One million salivary gland draining lymph node (DLN) cells or splenocytes were stained with PerCP-conjugated anti-mouse CD4 or APC-conjugated anti-mouse CD8 with PE-conjugated anti-mouse CD62L (BD Pharmingen, San Diego, CA, USA). Data were acquired using a FACSCalibur instrument (BD Biosciences, San Diego, CA, USA), and analyzed using Flowjo software (Tree Star Inc., Ashland, OR, USA). Flow cytometric identification of CD4+CD25+ Foxp3+ T regulatory (Treg) cells was performed as described previously [25] (link).

The same mice were used for isolation of naïve CD4⁺ T cells, which were purified from isolated autologous MNCs from EAE model mice using a CD4⁺ T cell magnetic bead separation kit (Catalog Number: 130-104-453, Miltenyi Biotec). The purity of the naïve CD4⁺ T cells was determined using peridinin chlorophyll protein (PerCP)-conjugated anti-mouse CD4 (Catalog Number: 550954), PE-conjugated anti-mouse CD25 (Catalog Number: 101904), and APC-conjugated anti-mouse CD127 MoAb (Catalog Number: 564175, BD Biosciences, San Jose, CA, USA) antibodies and a flow sorter, and only cells with purity > 90% were used for further experimentation; then, the naïve CD4⁺ T cells were further cocultured with the sCD40L-activated B cells to induce CD4⁺CD25⁺ Tregs.