Apc anti mouse cd274

Cells were digested using trypsin (Gibco, 25200056), washed with PBS (Hyclone, SH30256), and suspended in PBS. Tumor tissues from mice were carefully minced and digested using collagenase type IV (Gibco, 2049898) to get single tumor cells. Then, tumor cells were filtered with 70 μm Nylon mesh. After 600 × g centrifugation for 5 min, cells were resuspended in PBS. The cell suspension was incubated with APC anti-mouse CD274 (Biolegend, 124311) for 30 mins at 4°C. Flow cytometry analyses were performed on BD FACSCalibur (BD, USA).

Cells were stained for flow cytometry using CD3, CD4, and CD8 antibodies as well as dead cell marker for T-cells, and CD86, CD64, and CD11c for dendritic cells and macrophages. T-regulatory cells were fixed and stained using the T reg detection kit Miltenyi Biotec) according to manufacturer’s specification.
Antibodies used for flow were as follows: α-CD4 PE (Miltenyi Biotec), α-CD8 Vivobright FITC (Miltenyi Biotec), α-CD3 APC (MIltenyi Biotec), α-CD11c FITC (MiItenyi Biotec), α-CD86-PE (Miltenyi Biotec), and α-CD64-APC (Miltenyi Biotec).
Danger-Associated Molecular Patterns were analyzed by staining targeted cells with anti-human Calreticulin (CRT)-A647 (Bioss), anti-human HSP70-A647 (Bioss), anti-human GRP94-A647 (Bioss), and anti-human HMGB1-A647 (Bioss). yH2AX was analyzed by staining the cells using anti-human yH2AX (Biolegend). All antibodies were cross-reactive with mice.
To measure immunomarkers, the cells were stained with APC anti-mouse IFNAR-1 (Biolegend), APC anti-mouse CD252 (Biolegend), APC anti-mouse CD54 (Biolegend), APC anti-mouse CD152 (Biolegend), APC anti-mouse CD274 (Biolegend), APC anti-mouse CD137 (Biolegend), APC anti-mouse CEACAM-1/CD66a (R&D), Alexa Fluor 647 anti-mouse B7-H2 (R&D) Alexa Fluor 647 anti-mouse TRAILR1/TNFRSF10A (Novus), Alexa Fluor 647 anti-mouse CD27 ligand/TNFSF7/CD70 (Novus).
All cells were analyzed on a Guava Easycyte 8HT flow cytometer.