Pi rnase

The detection of cell proliferation and DNA content was performed using the baseclick EdU Flow Cytometry Kit (baseclick, Sigma-Aldrich, Arklow, Ireland) according to the manufacturer’s instructions. In brief, after 72 h exposure time, cells were incubated with 10 µM EdU (5-ethynyl-2′-deoxyuridine) for 2 h. Then, cells were harvested, centrifuged at 500× g for 5 min, washed with 1% BSA in PBS and fixed with the fixative solution. After a second wash with 1% BSA in PBS, cells were incubated with saponin-based permeabilization buffer in PBS before starting the click reaction. Cells were then washed with saponin-based permeabilization buffer and wash reagent, and incubated with a 10 µg/mL Propidium Iodide (PI)/100 µg/mL RNase solution in PBS (PI/RNase, Sigma-Aldrich, Arklow, Ireland) for 15 min at 4 °C. EdU and PI fluorescence was then measured using the CytoFLEX Flow Cytometer with a blue laser (488 nm) using FL-1 and FL-2 detectors. Data were analysed using CytExpert version 2.4.0.28 software (Beckman Coulter Inc., Indianapolis, IN, USA). For each assay, data of at least 10,000 events were collected.

HUVECs grown to 80% confluency were serum-starved in 1% FBS-containing M200 medium for 12 h, and treated with or without GDF15 for another 12 h. Following treatment, cells were released to enter the cell cycle with the addition of M200 complete medium containing 20% FBS for 12 h. The cell growth cycle was examined using propidium iodide (PI). 1.0 × 10⁶ cells were harvested by centrifugation, then fixed in ethanol and stained with PI/RNase (Sigma, USA). The samples were analyzed using FASC Calibur flow cytometer (Beckman Coulter Company, USA). Percentages of cells existing within the various phases of the cell cycle were calculated using Cell Quest by gating on G0/G1, S, and G2/M cell populations.