Click ittm plus edu flow cytometry assay kit

Cell proliferation was assessed using Click-iT^TM Plus EdU flow Cytometry Assay Kit (Thermo Scientific), according to the manufacturer’s instructions. Briefly, 10 µM 5-ethynyl-2’-deoxyuridine (EDU) was added to adherent subconfluent HUVEC cultures and cells were incubated in HUVEC medium for 24 h at 37°C. Cell cycle analysis was determined by flow cytometry with 488 nm excitation (Beckton Dickinson).

EdU and/or BrdU labelling was performed by injecting 2 mg of nucleoside analogue i.p. For the 16 h BrdU labelling time frame in Fig. 2c, d, mice were additionally maintained on 0.8 mg/ml BrdU-containing drinking water. BrdU incorporation was assessed using the eBioscience^TM BrdU Staining Kit for Flow Cytometry and a fluorochrome-conjugated antibody against BrdU (BU20A; Cat#11-5071-42), according to the manufacturer’s instructions. For concomitant EdU detection, the non-EdU cross-reactive anti-BrdU clone MoBU1 (Cat#MA1-12686) was used. EdU incorporation was analysed using the Click-iT^TM Plus EdU Flow Cytometry Assay Kit (ThermoFisher Scientific) prior to BrdU staining. Cellular DNA content was measured by staining samples with 7-Aminoactinomycin D (7-AAD; BD Biosciences) for 20 min at 4 °C immediately before acquisition. Apoptotic cells (fragmented/sub-G1 DNA content) were excluded from the analysis.

Cell proliferation was assessed using a Click-iT^TM Plus EdU flow Cytometry Assay Kit (Thermo Scientific), according to the manufacturer’s instructions. Briefly, 10 µM 5-ethynyl-2′-deoxyuridine (EdU) was added to adherent subconfluent HUVEC cultures at 72 h after siRNA transfection and cells were incubated in HUVEC medium for 24 h at 37 °C. Cell cycle analysis was determined by flow cytometry with 488 nm excitation (Beckton Dickinson).