Bv421 anti mouse cd3

Mice were euthanized, and single spleen cells were prepared. Spleen cells were then labeled with 1 μM CellTrace CFSE (Thermo Scientific) according to the manufacturer’s instructions. Cells in 96-well round-bottom plates were seeded at 0.5×10⁶/well using RPMI 1640 medium (Thermo Scientific), supplemented with 10% FBS (Gemini), 2 mM L-glutamine (Thermo Scientific), 100 U/mL penicillin (Thermo Scientific), 100 μg/mL streptomycin (Thermo Scientific), 10 mM HEPES (Thermo Scientific), and 50 µM 2-mercaptoethanol (Thermo Scientific), in the presence or absence of CII (50 μg/mL). The cells were cultured for 4 days, then stained with BV421 anti-mouse CD3 (BioLegend, 100228, [17A2], 1:100) and APC anti-mouse CD4 (BioLegend, 100411, [GK1.5], 1:100) after collecting the culture supernatant for cytokine measurement.

Alkaline Phosphatase (AP) anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG2c and IgG3 were acquired from Southern Biotech, while AP anti-mouse IgG was purchased from Bio-rad. BV421 anti-mouse I-A^b, PE anti-mouse I-A^d, BV421 anti-mouse H-2K^b, BV421 anti-mouse GL7, BV480 anti-mouse IgM, BV510 anti-mouse CD38, BB515 anti-mouse CD19, BV650 anti-mouse RORγt, PE CF594 anti-mouse GATA3, BB515 anti-mouse CD8α, and Alexa Fluor 647 (AF647) anti-mouse Bcl6 were obtained from BD Biosciences. FITC anti-mouse I-A^k, AF700 anti-mouse CD45, BV510 anti-mouse H-2K^d, PE anti-mouse H-2K^k, BV605 anti-mouse CD19, AF700 anti-mouse T cell receptor (TCR), AF700 anti-mouse CD11b, AF700 anti-mouse CD11c, BV650 anti-mouse IgD, BV421 anti-mouse CD3, BV510 anti-mouse CD4, BV605 anti-mouse Tbet, BV785 anti-mouse CD25, PerCP Cy5.5 anti-mouse CD19, PerCP Cy5.5 anti-mouse CD11c, PerCP Cy5.5 anti-mouse CD11b, and AF700 anti-mouse CD44 were purchased from Biolegend.