Cd4 phycoerythrin clone rpa t4

Single cell suspensions from TT specimens were resuspended in 100 µL of flow cytometry staining buffer (PBS with 1% FCS and 0.1% sodium azide). An FcR Blocking Reagent (Miltenyi Biotec) was used to block Fc receptors (FcR) and a 7-AAD viability dye (eBioscience, San Diego, CA, USA) was used to gate live cells. Cells were stained with surface antibodies against CD3-allophycocyanin-Cy7 (clone SK7, BD Pharmingen, San Jose, CA, USA), CD4-phycoerythrin (clone RPA-T4, BD Pharmingen), and CD8-fluorescein isothiocyanate (clone RPA-T8; BD Pharmingen). Cells were washed twice with flow cytometry staining buffer prior to re-suspension in Pre-Sort buffer (BD Biosciences, Oxford, UK). A BD FACSAria III SORP cell sorter on BD FACSDiva software (BD Biosciences) was used for sorting pure CD4⁺ (7AAD⁻CD3⁺CD4⁺CD8⁻), and CD8⁺ (7AAD⁻CD3⁺CD4⁻CD8⁺) TILs. Relevant procedures were followed to ensure minimum sorter-induced cell stress (SICS), as previously described [18 (link)]. Flow cytometric analyses were performed on FlowJo V10 software (FlowJo, Ashland, OH, USA).