FxCycle™ Far Red Stain

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	- Cells were incubated for 60 min in the presence of 10 μM 5-ethynyl-2’-deoxyuridine (EdU) at 37°C.

Protocol tips
- Cells were incubated for 60 min in the presence of 10 μM 5-ethynyl-2’-deoxyuridine (EdU) at 37°C.

Publication protocol

Cells cultured in the presence of ACY-241 and/or paclitaxel were incubated for 60 min in the presence of 10 μM 5-ethynyl-2’-deoxyuridine (EdU) at 37°C. After EdU incorporation, cells were washed and resuspended in fixative solution using the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Life Technologies, Grand Island, NY). Cellular DNA was stained using FxCycle™ Far Red Stain (Life Technologies) and cells analyzed on an Attune NxT Flow cytometer (ThermoFisher Scientific) or Cytomics FC 500 MPL flow cytometer (Beckman Coulter; Indianapolis, IN). Flow cytometry data were analyzed using FlowJo software (TreeStar; Ashland, OR). Dual-parameter plots were generated for Alexa Fluor® 488–labeled EdU fluorescence (indicating newly synthesized DNA) and FxCycle™ Far Red Stain fluorescence (indicating relative DNA content). The generated plots have a typical inverted U-shaped pattern that identifies proliferating cells with bright EdU staining and nonproliferating cells with dim EdU staining that are either in G1 phase (with 2N DNA content) or in G2/M phase (with 4N DNA content). Cell death is indicated by the subG1 population, which are the nonproliferating cells with dim EdU staining and low DNA content (< 2N; corresponding to the lower left corner of the dual-parameter plot).

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