Anti mouse cd8 percp cy5

The gastric mucosal tissue was cut into several small squares, ground into single-cell suspension, centrifuged, moistened with PBS, and stained with cell surface marker specific or isotype control antibody. After filtration with nylon mesh, it was analyzed by multicolor flow cytometry on FACSCanto II (BD Biosciences, San Jose, CA). The data were analyzed by FlowJo (Tree Star Inc.) or FACSDiva software (BD Biosciences).
The antibodies (BioLegend, San Diego, CA) were as follows: anti-mouse CD45-PE-Cy7 (stock no. 103113), anti-mouse CD11b-PerCP-Cy5.5 (stock no. 101227), anti-mouse Ly6G-FITC (stock no. 127605), anti-mouse Ly6C-PE (stock no. 128007), anti-mouse CD3-APC (stock no. 100235), anti-mouse CD8-PerCP-Cy5.5 (stock no. 100733), and anti-mouse CD4-PE (stock no. 100407).

Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Single-cell suspensions of digested tumors from OPC and OPC-API mice (1 × 10⁶ cells) were plated onto a 96 well plate coated with purified anti-mouse CD3 (10µg/mL) (Biolegend). Soluble purified anti-mouse CD28 (2µg/mL) (Biolegend) in RPMI 1640 media (10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) was added to the assay wells and then incubated at 37 °C in 5% CO₂. After two days of incubation, Golgi Plug, containing brefeldin A, (1 ug/mL) (BD Bioscience) was added to each assay well and incubated for 4 hrs. After incubation, cells were surface stained with fluorescent antibodies including anti-mouse CD3-FITC (Biolegend) and anti-mouse CD8-PercP/Cy5.5 (Biolegend). Samples were then intracellularly stained with anti-mouse IFN-γ-PE (eBioscience), along with isotype CTRL, using a Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer’s protocol. Acquisition of activated CD8⁺ T cell samples were performed using a flow cytometer BD LSRII (BD Biosciences Immunocytometry Systems). FlowJo software (BD Bioscience) was used for data analysis.