Anti foxp3 nrrf 30

Draining LN cells (axillary, brachial, and inguinal) were incubated in 0.2 mM CFSE for 8 min at room temperature (Life Technologies). CFSE-labeled cells were washed and cultured in medium only, with rhMOG, or with OVA protein (Sigma, St. Louis, MO) at 10 or 30 mg/ml for 96 h. Culture supernatants were collected for cytokine measurement, and cells were restimulated with 13 cell stimulation mixture (eBioscience) in the presence of 3 mg/ml brefeldin A (eBioscience) for 4 h. Surface Ags were stained with anti-TCRb, anti-CD4, and anti-CD19, and followed by the intracellular staining. For the detection of Foxp3 and intracellular cytokines, cells were treated with Foxp3 Fixation/Permeabilization buffer (eBioscience) and stained with anti-Foxp3 (NRRF-30; eBioscience) and anti–IL-17A (TC11-18H10.1; Biolegend) as an Ab pair or anti–IFN-g (XMG1.2; BD Biosciences) and anti–IL-17A as a pair in the FACS buffer.

Rat anti-mouse CD40 mAb (FGK4.5), rat IgG2a (2A3) and rat IgG2b (LTF-2) isotype controls were purchased from BioXcell (West Lebanon, NH). Rat anti-CD4 mAb (GK1.5) was purchased from Taconic (Hudson, NY). The following antibodies were purchased from BD Biosciences (San Jose, CA): anti-CD80 (16-10A1), anti-CD86 (GL1), anti-CD45 (30-F11), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5) and anti-CD25 (PC61). The following antibodies were purchased from eBioscience (San Diego, CA): anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53–6.7), anti-IFN-γ (XMG1.2) and anti-Foxp3 (NRRF-30).