Anti mouse cd4 mab

Crude wheat gluten and ovalbumin (OVA) were obtained from Sigma (Sigma, St. Louis, MO), while gliadin was from Fluka (Sigma). Phorbol myristate acetate (PMA) and ionomycin were purchased from Sigma. The following monoclonal antibodies (mAbs) as well as isotype controls were purchased from BD Biosciences (BD Biosciences, Mountain View, CA): Alexa Fluor 488-conjugated rat anti-mouse IL-2 (JES6-5H4, IgG_2b), IL-4 (11B11, IgG₁), IFN-γ (XMG1.2, IgG₁), FITC-conjugated rat anti-mouse IL-10 (JES5-16E3, IgG_2b) and CD8 (53-6.7; IgG2a,κ), PerCP-Cy5.5-conjugated hamster anti-mouse CD3 (145-2C11; IgG1,κ), rat anti-mouse CD4 (RM4-5; IgG2a,κ), CD8 (53-6.7; IgG2a,κ) and PE-conjugated hamster anti-mouse γδ T cell receptor (GL3; IgG2,κ) mAbs. Mouse Treg staining kit Cat.No. 88–8111, PE-conjugated rat anti-mouse Foxp3 mAb (FJK-16s; IgG2a,κ) and FITC-conjugated rat anti-mouse CD4 mAb (RM4-5; IgG2a,κ) were from eBioscience (eBioscience, San Diego, CA). The anti-mouse CD4 mAb (BD Biosciences) was used in combination with intracellular cytokine staining using Cytofix/Cytoperm kit (BD Biosciences), while the anti-mouse CD4 mAb (from the eBioscience kit no. 88–8111) was used when detecting Foxp3⁺CD4⁺ T cells (with no prior PMA inomycin stimulation) by using the Treg staining kit 88–8111.

Single-cell suspensions were stained with relevant fluorochrome-conjugated anti-mouse/human CD11b, anti-mouse Gr-1, Ly6G, and Ly6C antibodies (Biolegend, San Diego, CA) and anti-human-CD33 and HLA-DR antibodies (eBioscience, San Diego, CA). To examine cytotoxic T lymphocytes (CTLs) and T helper 1 (Th1) cells, single-cell suspensions derived from tumor tissues of tumor-bearing mice were treated with 1 μg/mL ionomycin, 2 ng/mL monensin (eBioscience, San Diego, CA), and 50 ng/mL PMA (Sigma-Aldrich, St. Louis, MO) for 5 h. After resuspending in PBS, the cells were stained with anti-mouse CD3e, anti-mouse CD8a or anti-mouse CD4 mAb (eBioscience, San Diego, CA). The cells were incubated at 4°C for 30 min, fixed, permeabilized, and stained with anti-mouse IFN-γ mAbs (BD Pharmingen™) according to the instructions in the intracellular cytokine staining kit (eBioscience, San Diego, CA). Flow cytometry (BD FACSCalibur) was used to determine the proportion of cells.