Cd4 pe clone rpa t4

Following phosphate-buffered saline (PBS) washing, single cell suspension from tumor tissue was washed and resuspended in 100 µL of flow cytometry staining buffer (PBS with 1% fetal calf serum (FCS) and 0.1% sodium azide). Fc receptor (FcR) Blocking Reagent, human (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to block FcR. Cells were stained with cell surface antibodies against CD3-APC-Cy7 (clone SK7, BD Pharmingen, San Jose, USA), CD4-PE (clone RPA-T4, BD Pharmingen), CD8-FITC (clone RPA-T8; BD Pharmingen), CD33-APC (clone WM53, BD Pharmingen) and 7-AAD viability dye (eBioscience, San Diego, USA) to exclude dead cells and gate on live cells.
Cells were washed with flow cytometry staining buffer then re-suspended in Pre-Sort buffer (BD Biosciences). BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD8⁺ (7AAD^–CD3⁺CD4^–CD8⁺CD33^–), CD4⁺ (7AAD^–CD3⁺CD4⁺CD8^–CD33^–) and CD33⁺(7AAD^–CD3^–CD4^–CD8^–CD33⁺) populations. The sorting strategy is shown in figure 1A. We used stringent gating strategy and applicable measures to ensure minimal sorter-induced cell stress. High purities of sorted immune cell populations were always checked and confirmed. FlowJo V.10 software (FlowJo, Ashland, USA) was used for data analyzes.