Anti gr 1 pe

Single-cell suspensions were generated from spleen and liver of tumor-bearing mice. The following anti-mouse antibodies were used: anti-CD45-BUV395, anti-CD3E-PE/CF594, anti-CD44-PE, anti-CD4-APC/H7, anti-CD8-APC, anti-CD69-FITC, anti-CD86-FITC, anti-CD11c-PE/Cy7, anti-Gr1-PE, anti-CD11b-APC, anti-CD19-PE/Cy7, anti-NK1.1-PerCP/Cy5.5 (BD Bioscience, San Diego, CA), anti-Ly6C-BV421, anti-Ly6G-AF700, anti-CD4-FITC, anti-CD62L-PerCP/Cy5.5, anti-NKG2D-FITC, anti-F4/80-PerCP/Cy5.5, anti-IFN-γ-PE and anti-TNF-α-PE/Cy7 (Biolegend, San Diego, CA). Anti-mouse CD16/32 FcR block antibody was purchased from Biolegend (San Diego, CA). For intracellular cytokine staining, cells were stimulated with Cell Activation Cocktail (Biolegend, San Diego, CA) for 4 hours and stained with surface antibodies. Then cells were fixed, permeabilized and stained with intracellular cytokine antibodies. Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA).^{29 (link)} Data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Immunofluorescence staining of slides was performed as described for immunohistochemistry until antigen retrieval. Then, slides were treated with or without 1% Triton X-100 for 20 min, followed by blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, slides were incubated with anti-CD3e-FITC (BD), anti-CD8a-PE (BD), anti-CD4-PE (BD), anti-CD11b-FITC (BD), anti-Gr-1-PE (BD), anti-Foxp3-PE (BD), and anti-IL-17A-Alexa Fluor 647 (BD) for 30 min at room temperature. Slides were then washed three times with PBS-T, incubated with Hoechst-33342 for 5 min at room temperature, washed again with PBS-T three times, and sealed with glycerin.

The spleens of mice were placed in cold RPMI-1640 medium immediately and then cut into small pieces with eye scissors. Single-cell suspensions of spleen tissues were acquired after filtration with 300- and 100-mesh filters. Single-cell suspensions of bone marrow tissues were obtained from the thigh bones of mice after being washed with cold PBS. The red blood cells were then disrupted within the cell suspension. For treatment, 2×10⁶ cells were stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA; 50 ng/ml) plus 2 µg/ml ionomycin (Sigma) and monensin (Sigma; 5 µg/ml) for 5 h. The cells were then collected for Th17 cytokine staining. Subsequently, they were fixed and/or permeabilized using fixation/permeabilization kits (BD, USA) according to the manufacturer's instructions, and incubated with anti-interleukin (IL)-17A-Alexa Fluor (BD), anti-CD4-PE (BD), anti-CD4-APC (BD), anti-CD25-PE-CyTM7 (BD), anti-FoxP3-PE (eBioscience, San Diego, CA, USA), anti-CD3e-FITC (BD), anti-CD4-PE-CyTM⁷ (BD), anti-CD8-PE (BD), anti-CD11b-APC (BD), and anti-Gr-1-PE (BD) antibodies for 40 min at 4°C. The cells were incubated with rat anti-mouse CD16/CD32 (BD) for 30 min before CD11b and Gr-1 staining at 4°C. Flow cytometry (BD) was performed, and data were analyzed using FlowJo software.