Melody flow cytometer

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.

Approximately 1 × 10⁵ THY1⁺ cells were collected and fixed at 4 °C with cold ethanol for at least 2 h. The cells were then pelleted by centrifugation at 400 × g for 5 min and resuspended in cold PBS, and this process was repeated. Cells were resuspended in propidium iodide (PI) staining buffer (20 μg/ml PI, 0.5 mM EDTA, 0.5% NP40, 0.2 mg/ml RNase An in PBS). Cells were incubated in PI staining buffer for 2 h at 37 °C in the dark. After incubation, PI fluorescence was analyzed on a Melody flow cytometer (BD Bioscience) to determine the percentage of cells in each stage of the cell cycle with FlowJo software.