Anti b7h1 phycoerythrin pe

Expression of PD-L1 was assessed using anti-B7H1-phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA) at a concentration of 0.05 μg/ml. A relative Ig isotype PE-conjugated antibody was used as a control of non-specific binding. Samples were analysed using a BD Accuri™ C6 Cytometer (BD Biosciences, New Jersey, USA). Analysis of apoptosis was performed by annexin-V staining in double fluorescence with CD45-Peridinin Chlorophyll Protein Complex (PerCP) conjugated. Briefly, 1 × 10⁵ cells were harvested 6 h after co-culture (see co-culture paragraph) and resuspended in 100 μl of binding buffer (10 μM Hepes/NaOH pH 7.5, 140 μM NaCl, and 2.5 μM CaCl₂) containing 1 μl of annexin-V-FITC (Pharmingen/Becton Dickinson, San Diego, CA, USA) and 5 μl of CD45 for 15 min at room temperature in the dark. Then, 100 μl of the same buffer was added to each sample and analysed with BD Accuri™ C6 Cytometer (BD Biosciences).

Expression of PD-L1 was assessed using anti-B7H1-phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA) at a concentration of 0.05 μg/ml. A PE-conjugated control Ig isotype was used to assess non-specific binding. Briefly, cells were harvested and incubated with the antibodies mentioned above for 30 min in the dark at 4 °C, washed and then analyzed with a BD Accuri™ C6 Cytometer (BD Biosciences, New Jersey, USA). Cleaved caspase-3 immunostaining was performed as previously described^{23 (link)}. Apoptosis was assessed by two methods: propidium iodide (PI) incorporation to analyze the DNA content in permeabilized cells^{24 (link)}, and the annexin V-binding assay in double staining with PI, according to Vermes et al.^{25 (link)}. Cells were analyzed with a BD Accuri™ C6 Cytometer (BD, Becton Dickinson).

PD-L1 expression was measured using anti-B7H1-phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA) at a concentration of 0.05 μg/mL. A PE-conjugated control Ig isotype was used as control for non-specific binding. Briefly, cells were harvested and incubated with the antibodies for 30 min in the dark at 4 °C, washed and then analyzed by a BD Accuri™ C6 Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).