Fxcycle far red dye

To quantify the cells in each cell cycle phase, Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (C10420, Thermo Fisher) was used according to the manufacture’s instruction. In brief, cells were incubated with 25 μM 5-ethynyl-20-deoxyuridine (EdU) for one hour and collected using Cell Dissociation Buffer (Gibco) following a PBS wash. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with 1% BSA in PBS. Cells were then permeabilized for 15 min with saponin-based permeabilization/wash buffer and incubated with the Click-iT reaction cocktail for 30 min protected from light. Cells were washed once with saponin-based permeabilization/wash buffer and stained for DNA content using the FxCycle Far Red dye (Invitrogen). Cells were analyzed with Fortessa cell analyzer (BD) and FlowJo software.

Cell cycle profile analysis was performed using the Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In summary, cultured cells were incubated at 37°C with 10 μM EdU (5-ethynyl-2′-deoxyuridine) for 1 hr and harvested using cell dissociation buffer (Gibco). After three washes with PBS/1% BSA, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three more times with PBS/1% BSA. Cells were then permeabilized for 15 min with saponin-based permeabilization/wash buffer and incubated with the Click-iT reaction cocktail for 30 min protected from light. Cells were washed once with saponin-based permeabilization/wash buffer and stained for DNA content using the FxCycle Far Red dye (Invitrogen). Cells were analyzed on the Cyan ADP flow cytometer and FlowJo software.