Anti cd45ro apc

After 24, 48, 72, and 120 h-cultures, cells were washed twice in sorter buffer (PBS containing 1% FCS [Gibco BRL, USA] and 0.1% sodium azide [Sigma]) and blocked for 10 min with PBS containing FC-receptor bloking solution (BioLegend Inc., San Diego, CA, USA), then stained for 30 min in the dark with fluorochrome-conjugated monoclonal antibodies (mAbs) against the relevant cell surface antigens to characterize main leukocyte populations (anti-CD3 FITC, PE, and PerCP; anti-CD4 PerCP and APC; anti-CD8 PerCP; anti-CD14 FITC; anti-CD83 APC; anti-CD80 PerCP; and anti-CD86 PE) and cell activation status (anti-CD69 PE and Cy and anti-TLR2 APC) and to identify T cell phenotypes as naïve, memory, or effector (anti-CD45RO APC and anti-CD62L FITC). All mAbs were obtained from Becton Dickinson (BD Biosciences). After incubation at 4°C, cells were washed twice for 10 min, first with sorter buffer and then with PBS containing 0.1% sodium azide. Cells were then fixed with 1% paraformaldehyde (Sigma) and stored at 4°C in the dark. Cells were collected within 48 h on the FACSAria flow cytometer (BD Biosciences). Isotype controls were used to define the negative cell region, and 50,000 events were acquired for each sample. The data were analyzed using FlowJo version 8.2 software (Treestar, Ashland, OR, USA).

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.