Anti cd14 and anti cd11b macs magnetic sorting system

Ovarian biopsy specimens (n=31) were cut into small pieces and digested in RPMI 1640 (Gibco, USA) supplemented with 0.25% trypsin (Gibco), 0.002% DNaseI (Gibco), and 20% fetal bovine serum (FBS, Gibco) at 37°C for 20 min. TILs within dissociated cells were filtered through a 100μm mesh and isolated by Ficoll-Hypaque density gradient centrifugation (Beijing Chemical Reagent Company, China). The mononuclear cells were washed and resuspended in medium supplemented with 10% heat-inactivated FBS for FACS analysis. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation, sequentially using the anti-CD14 and anti-CD11b MACS magnetic sorting system (Miltenyi Biotec, Germany) within 2h of sample collection. PMN-MDSCs (CD14⁻CD11b⁺) and M-MDSCs (CD14⁺CD11b⁺) were enriched according to the manufacturer’s instructions. The purity of the two subsets of cells was >95%. CD8⁺ T cells were further purified by MACS magnetic sorting system according to the manufacturer’s protocol (MiltenyiBiotec, Auburn, CA, USA) and the purity was also >95%. Then, CD8⁺ T lymphocytes were magnetically isolated, as confirmed by flow cytometry.