Lsr dual fortessa flow cytometer

To measure DNA replication and cell cycle stage, EdU (5-ethynyl-2´-deoxyuridine) was added to cells at 10 nM for 1.5 h before harvesting. 1 confluent well of a 6-well plate of HCT116 cells were harvested and processed as per manufacturer’s instructions for the Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Thermo Fisher cat: C10634). Per manufacturer’s instructions, FxCycle Violet DNA content stain (Thermo Fisher cat: F10347) was added after the Click-iT reaction at 1:1000 dilution before quantifying on a BD LSR Dual Fortessa flow cytometer. Alexa Fluor 647 was measured in the 670-30 Red C-A Channel and FxCycle Violet Stain was measured in the 450-50 Violet F-A Channel. Analysis was performed using FACS DIVA and FlowJo V10 (FlowJo, LLC) software.

To measure DNA replication and cell cycle stage, EdU (5-ethynyl-2´-deoxyuridine) was added to cells at 10nM for 1.5 hrs before harvesting. 1 confluent well of a 6-well plate of HCT116 cells were harvested and processed as per manufacturer’s instructions for the Click-iT^™ Plus EdU Alexa Fluor^™ 647 Flow Cytometry Assay Kit (Thermo Fisher cat: C10634). Per manufacturer’s instructions, FxCycle Violet DNA content stain (Thermo Fisher cat: F10347) was added after the Click-iT reaction at 1:1,000 dilution before quantifying on a BD LSR Dual Fortessa flow cytometer. Alexa Fluor 647 was measured in the 670–30 Red C-A Channel and FxCycle Violet Stain was measured in the 450–50 Violet F-A Channel. Analysis was performed using FACS DIVA and FlowJo V10 (FlowJo, LLC) software.