Click it flow cytometry kit

To study cell viability, hCPC were detached with trypsin–EDTA 48 h post-transfection, labeled with DAPI (1/1000; Sigma-Aldrich) and quantified by flow cytometry on a FACS Canto 3L flow cytometer (BD Biosciences, San Jose, CA). For proliferation assays, 5-ethynyl-2’-deoxyuridine (EdU; 10 μM) was added to hCPC cultures 12 h prior to analysis. Proliferating cells were detected with the Click-iT Flow-Cytometry Kit (Thermo Fisher Scientific). For apoptosis analysis, cells were exposed to H₂O₂ (500 μM, during 5 h), then collected (including detached cells) and labeled at 4ºC for 15 min with AnnexinV-FITC (diluted 1:10) in the binding buffer provided by the manufacturer (ApoScreen® Annexin V Apoptosis Kit-FITC; Southern Biotech, Birmingham, AL). Labeled cells were washed with PBS/0.01% BSA and resuspended in 390 μL of binding buffer. Propidium iodide (50 μg/ml, Beckman Couler, Nyon, Switzerland) was added (1:40 dilution) for dual-staining and cells were analyzed by flow cytometry. DAPI and AnexinV/PI positive-cells were quantified on a FACS Canto 3L flow cytometer (BD Biosciences). When indicated, necrosis of hCPC was induced by a short heat treatment (10 min, 60ºC) of attached monolayers.