Muse® Cell Cycle Assay Kit

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	- Cells were treated with different concentrations of 5‘-Cl (0.0 to 8.0 µM) for 24h or with 4.0 µM for various periods of time (0 to 48h). - Cells were fixed in ice-cold 70% ethanol at -20°C for 3h. - Stained with 200 µL of PI/RNAse reagent for 30 min.

Protocol tips
- Cells were treated with different concentrations of 5‘-Cl (0.0 to 8.0 µM) for 24h or with 4.0 µM for various periods of time (0 to 48h). - Cells were fixed in ice-cold 70% ethanol at -20°C for 3h. - Stained with 200 µL of PI/RNAse reagent for 30 min.

Publication protocol

Distribution of cells in different stages of cell cycle was analyzed by flow cytometry using the Muse™ Cell Cycle Kit from EMD Millipore Bioscience (Darmstadt, Germany). The kit utilizes propidium iodide (PI) staining to allow quantitative measurements of percentage of cells in the G0/G1, S and G2/M phases on the Muse™ Cell Analyzer (EMD Millipore Bioscience). The cell cycle analysis was performed according to the manufacturer's protocol, and as recently described [31]. Briefly, exponentially growing HL-60 cells (5 × 105 cells/mL) were treated with different concentrations of 5‘-Cl (0.0 to 8.0 µM) for 24h or with 4.0 µM for various periods of time (0 to 48h). After incubation, 1 × 106 cells were harvested and washed with PBS, followed by fixation with ice-cold 70% ethanol at -20°C for 3h. The cells were then washed with PBS, stained with 200 µL of PI/RNAse reagent for 30 min and analyzed by the Muse™ Cell Analyzer. Adherent cells were trypsinized after treatment, washed once with PBS, and fixed in 70% ethanol overnight at -20°C before analysis as described above. All the compound stock solutions were prepared in a solvent containing 50% DMSO and 50% PBS. Therefore, the control cells were treated with equal amount of the carrier solvent (0.05% DMS0), and showed no effect on cell cycle when compared with the solvent-free control cells. Under similar conditions, cells were treated with 0.02 µM and 0.04 µM cytarabine as internal controls to validate the assay results (data not shown).

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