BD Cycletest™ Plus DNA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Do not vortex while adding and mixing of solutions A, B and C

Protocol tips
Do not vortex while adding and mixing of solutions A, B and C

Publication protocol

Flow Cytometry Analyses
Flow cytometry was used to evaluate the cell cycle profile, apoptosis, and mitochondrial membrane potential of FKB-treated HeLa cells. The cell cycle profile of untreated controls and HeLa cells treated with FKB for 48 hours was evaluated using BD CycletestTM Plus DNA kit according to the manufacturer’s protocol. Briefly, harvested control and treated HeLa cells were incubated with 250 µL of Solution A (10 minutes), followed by 200 µL of trypsin inhibitor and RNase buffer Solution B (10 minutes), and finally by 200 µL of propidium iodide (PI; 10 minutes). The samples were then analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). For apoptosis quantification, BD Annexin-V/PI apoptosis kit (BD, USA) was used according to the manufacturer’s protocol. In brief, harvested control and FKB-treated HeLa cells were incubated with 5 µL of AnnexinV-FITC and 5 µL of PI for 15 minutes. Then, the samples were topped up with 400 µL of binding buffer and analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). BD MitoScreen JC-1 kit was used for quantification of mitochondrial membrane potential. In brief, harvested control and FKB-treated HeLa cells were incubated with 500 µL of JC-1 working solution (15 minutes), washed, and resuspended with 500 µL of assay buffer. The samples were then analyzed by BD FACS Calibur using BD CellQuest Pro software (BD, USA).

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Manufacturer protocol

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