Facsaria 3 sorp cell sorter on facsdiva software

Cells were stained with cell-surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen) and CD33-allophycocyanin (clone WM53, BD Pharmingen). Cells were washed twice with flow cytometry staining buffer and re-suspended in Pre-Sort buffer (BD Biosciences). 7-AAD viability dye (eBioscience, San Diego, USA) was used to gate live cells. BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4⁺ (7AAD^–CD3⁺CD4⁺CD8^–CD33^–), CD8⁺ (7AAD^–CD3⁺CD4^–CD8⁺CD33^–) and CD33⁺(7AAD^–CD3^–CD4^–CD8^–CD33⁺) populations. Applicable measures were taken to ensure minimal sorter-induced cell stress (SICS). Data analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).

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.