Anti pd 1 monoclonal antibody clone rmp1 14

Tumor cells were harvested by trypsinization, washed three times with PBS, and injected subcutaneously into the right flank of recipient mice at 10⁵ to 10⁶ cells in 100 μl of endotoxin-free PBS. Tumor cells were >98% viable at the time of injection as determined by propidium iodide staining. Tumor size was quantified as the mean of the longest diameter and its perpendicular. For COX inhibition in vivo, aspirin was administered in the drinking water at 600 μg/ml 1 to 3 days before injection of tumor cells and replaced every 3 days. Alternatively, mice received 200 μl of celecoxib (Sigma and LC Laboratories) intraperitoneally (i.p.) at 500 μg/ml (12.5% DMSO in PBS) daily from day 0. Anti-PD-1 monoclonal antibody (clone RMP1-14, BioXCell) was administered i.p. at 200 μg/mouse from day 3 post-tumor cell inoculation every 3 to 4 days for a maximum of six injections.

Anti-PD1 monoclonal antibody (clone: RMP1-14) and an isotype control were purchased from BioXCell (West Lebanon, NH, USA) and stored at 4°C. Cilengitide was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Recombinant mouse interleukin-6 (IL-6, HY-P7063) and recombinant human IL-6 (HY-P7044) were purchased from MedChemExpress, dissolved in phosphate buffered saline (PBS, Promotor, Wuhan, Hubei, China) and stored at −20°C.