Muse® Cell Cycle Assay Kit

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	- Cells were treated with DMSO, 10 μM NAC, or 10 μM pifithrin-β; - cells were pre-exposed to 10 μM pifithrin-β for 3 h and then supplemented with 10 μM NAC. - Cells are fixed with 70% ethanol at -20°C for overnight.

Protocol tips
- Cells were treated with DMSO, 10 μM NAC, or 10 μM pifithrin-β; - cells were pre-exposed to 10 μM pifithrin-β for 3 h and then supplemented with 10 μM NAC. - Cells are fixed with 70% ethanol at -20°C for overnight.

Publication protocol

The measurement of the percentage of cells in
the G0/G1, S, and G2/M phases of the cell cycle was performed using the Muse Cell Analyzer, as previously reported.40 Briefly, cells were treated with DMSO, 10 μM NAC, or 10 μM pifithrin-β; in parallel, cells were pre-exposed to 10 μM pifithrin-β for 3 h and then supplemented with 10 μM NAC. After 24 h, adherent cells were collected and centrifuged at 300g for 5 min. After being washed with 1× PBS, suspended cells were fixed and, after the slow addition of 1 mL of ice-cold 70% ethanol, maintained overnight at −20 °C. Then, a cell suspension aliquot (containing at least 2 × 105 cells) was centrifuged at 300g for 5 min, washed once with 1× PBS, and suspended in the fluorescent reagent (Muse Cell Cycle reagent). After incubation for 30 min at room temperature, in the dark, the measurements of the percentages of cells in the phases of the cell cycle were acquired.

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