Anti cd8 fitc

Single-cell suspensions of thymocytes were stained for 20 min with anti-CD4-PE, anti-CD8-FITC, or anti-TCR-β-APC antibodies (1:50; Miltenyi Biotec, Bologna, Italy) at 4 °C. Dead cells were stained with SYTOX® Blue (Thermo Fisher Scientific-Life Technologies, Waltham, MA, USA). Analysis was performed with CyAn cytofluorimeter (Dako, Milan, Italy) and analyzed using FlowJo software (version 7.2.4). Blood was collected from mouse eyes 1 week before the sacrifice and cells stained with anti-CD45-eFluor450 antibody (1:100; eBioscence, San Diego, CA, USA) to exclude red cells and with anti-CD3-APC, anti-CD4-PE, and anti-CD8-FITC antibodies (1:50; Miltenyi Biotec) to detect mature T cells.

After 22 h-incubation of 5×10³ PC3-PSCA tumor cells with 5×10⁴ freshly isolated PBMCs or isolated CD8+ T cells in the presence or absence of 30 pmol/ml of recombinant Abs, cells were spun down and supernatants were collected. T cells of one triplet were pooled, stained with anti-CD25/PE, anti-CD69/PE-Cy5, and anti-CD8/FITC (Miltenyi Biotec GmbH) and measured using a flow cytometer. Cytokine concentrations in collected supernatants were determined using OptEIA Human IFN-γ and OptEIA Human TNF ELISA Kits (BD Biosciences).

Flow cytometry was performed using a NovoCyte flow cytometer (ACEA Biosciences, Inc., San Diego, CA, USA). For PD1 expression analyses, mGFP or PD1-mGFP transduced MSCs were labeled with anti-PD1-APC antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). For CD107a expression analysis, HuCCT1 cells were pretreated with 1 × 10¹⁰ control CDNVs and PD1+ CDNVs for 3 h, followed by co-culture with T cells or NK cells for 6 h at a 5:1 or 10:1 effector to target (E:T, immune cell: HuCCT1 cell) ratio in the presence of GolgiStop (BD Bioscience, Franklin Lakes, NJ, USA) and anti-CD-107a-APC antibody (BD Bioscience, Franklin Lakes, NJ, USA) in RPMI 1640 medium. The immune cells were collected and stained with anti-CD8-FITC or anti-CD56-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for NK and T cells, respectively, prior to flow cytometry. Cytometry data were analyzed using FlowJo v10.8.1.