Anti cd45ra pe

For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.

Hypaque-1077 (Sigma-Aldrich, Gillingham, UK) was used to isolate peripheral blood mononuclear cells (PBMCs) of SCLC patients and healthy controls. The isolated cells were re-suspended in RPMI-1640 medium (Biosera, Heathfield, UK), supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Solarbio, Beijing, China). PBMCs were frozen in RPMI-1640 medium supplemented with 20% FBS and 10% DMSO (Sigma-Aldrich, Gillingham, UK) and stored at −80 °C until flow cytometric analysis.
PBMCs were stained for the expression of surface markers using the following anti-human fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-Cy7; anti-CD4 BV510; anti-CD8 APC-Cy7; anti-CD45RA PE; anti-CD45RO APC; anti-CCR7 FITC; anti-PD-1 PerCPCy5.5; and anti-PD-L1 BV421 (all antibodies were purchased from Biolegend, San Diego, CA, USA). Staining was performed in FACS buffer, 1% PBS-BSA, for 30 min, on ice, in the dark. Acquisition and multicolor analysis were performed using BD FACSChorus Software version 3.0. on a Melody flow cytometer (BD Biosciences, Heidelberg, Germany). For T-cell subsets, the analysis gates were restricted to the lymphocytic population. Each measurement contained 10⁶ single events. Unstained cells were used as negative control, whereas FMO-stained cells were used in order to set the gates.