Fitc conjugated gr 1 antibody

For the isolation of myeloid-derived suppressor cells (MDSCs), 1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 20 days later, splenocytes were collected from naïve (control) or tumor-bearing mice and prepared for flow cytometry analysis to measure CD11b and Gr-1 positive cells. Splenocytes were stained with FITC-conjugated Gr-1 antibody (BD Pharmingen, San Diego, CA) and PE-CD11b antibody. MDSCs were purified by magnetic cell sorting using the mouse CD11b MicroBeads according to the manufacturer's instructions (MACS; Miltenyi Biotec, Auburn, CA). The purity of CD11b⁺ cells obtained by this method was 90–95% and 90% of CD11b⁺ cells were Gr-1⁺ as determined by flow cytometry. For the detection of MDSCs in tumor tissue, TC-1 tumor tissues obtained from tumor-bearing mice were cut into fragments in phosphate-buffered saline (PBS), washed twice with PBS, and then digested with 500 U/ml of Dispase (Godo Shusei, Co., Ltd. Tokyo) at 37°C for 20 min. The supernatants of the digestion were discarded and the remaining fragments were suspended into 5 ml of PBS. The cell suspensions were passed through a stainless wire sieve (100 mesh), washed twice with 20 ml of PBS and centrifuged for 5 min at 15O×g. Pelleted cells were resuspended in PBS and stained with FITC-conjugated Gr-1 antibody (BD Pharmingen, San Diego, CA) and PE-CD11b antibody.