Apc conjugated anti mouse cd4

Mouse splenocytes were collected aseptically from spleens of mice by mincing the spleen tissues in a sterile Petri plate, and the erythrocytes were lysed in lysis buffer (10 mmol/L KHCO₃, 150 mmol/L NH₄Cl, 10 mmol/L ethylenediaminetetraacetic acid, pH 7.4). Splenocytes were prepared from tumor-grafted mice or naïve mice. ACK (Ammonium–Chloride–Potassium) Lysing buffer was used to lyse erythrocytes. Splenocytes were stained using APC-conjugated anti-mouse CD3e antibody, APC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8a, APC/Cy7-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, and PE-conjugated anti-mouse Ly6G/Ly6c (Gr-1) (1:200, BioLegend, San Diego, CA, USA) following the manufacturer’s instructions. Living cells were assessed by using 1 mg/mL Propidium iodide (PI). Analyses of fluorescence staining were performed with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Data were collected and analyzed using Kaluza^® for Gallios-Acquisition and Flow Analysis Software (Beckman Coulter). We collected at least 10,000 viable cell events per sample in each experiment.

Cells were incubated with specific antibodies for 15 min at 4 °C in the dark for cell surface staining. For intracellular staining, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μg/ml ionomycin (MCE) in the presence of GolgiStop (BD Biosciences) for 4 h. Subsequently, cells were fixed and permeabilized with BD Cytofix/Cytoperm Plus (BD Biosciences), and incubated with specific antibodies for another 30 min at 4 °C in the dark. All samples were acquired with FACSVerse flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). The following antibodies were used: PE-CY7-conjugated anti-mouse F4/80 (BioLegend), PerCP-conjugated anti-mouse CD11b (BioLegend), APC-CY7-conjugated anti-mouse CD45RA (BioLegend), PerCP-conjugated anti-mouse CD3 (BioLegend), PE-conjugated anti-mouse CD4 (BioLegend), APC-conjugated anti-mouse CD4 (BioLegend), Bv421-conjugated anti-mouse IL-17A (BioLegend), PE-conjugated anti-mouse IL-17A (BioLegend).

The expression of CD3, CD4, CD8a in the TEM were measured by flow cytometry. Tumor tissues were harvested after injection and digested with Collagenase II (1 mg/ml, Sigma-Aldrich, C685, Pudong, Shanghai, China) in DMEM, which contained DNase (0.3mg/ml, Sigma-Aldrich, DN25) at 37°C with 800 rpm for 1 h. Afterward, the cell suspensions were filtered through a 70 µm strainer. Single-cell suspensions were stained with PerCP/Cyanine5.5 conjugated anti-mouse CD3 (Cat # 100217, clone 17A2, Biolegend), PE-conjugated anti-mouse CD8a (Cat #100707, clone 53-6.7, Biolegend), and APC conjugated anti-mouse CD4 (Cat #100411, clone GK15, Biolegend) for 30 min on ice, following the manufacturer’s instructions. Samples were analyzed by flow cytometry system and the data were analyzed by FlowJo software (Ashland, OR, USA).