Flow cytometry analysis was performed to determine any disruption during the cell cycle of HCT-116 cells in the presence of Dis. HCT-116at density of 2 × 106 cells were seeded and treated for 24 h, 48 h and 72 h in a 75 cm2 flask. Negative control was treated with 0.02% DMSO. After fixation of the cells with 70% ethanol, cells were preserved in −20 °C overnight followed by washing and staining with 500 µl of PI/Rnase dye (BD Biosciences, San Jose, CA, USA) and incubated for 30 min. DNA content analysis was performed using BD FACSCanto II flow cytometer instrument and BD FACS Diva software (BD FACSCanto™II, San Jose, CA, USA). A total of 15,000 events per sample were recorded for analysis. ModFit LT version 3.0 software was used for analyzing the data.
Pi rnase dye
Manufactured by BD
Sourced in United States
PI/RNase dye is a fluorescent staining solution used for DNA content analysis. It binds to DNA and allows for the measurement of the DNA content in cells. The dye is designed to selectively stain the DNA in the presence of RNA, providing a precise assessment of the DNA content within a sample.
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2 protocols using pi rnase dye
1
Cell Cycle Analysis of HCT-116 Cells
2
Cell Cycle and Apoptosis Assessment
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To assess the effect of gene expression on the cell cycle, cells were centrifuged, fixed with ethanol, and washed with PBS; 500 µL of PI/RNase dye (BD Biosciences, USA) was then added. The cells were incubated in darkness for 15 min, and then the cells were analyzed by flow cytometry (BD Accuri C6 Plus; BD Biosciences, USA).
To assess the effect of gene expression on cell apoptosis, cells were centrifuged and resuspended in 50 µL 1× binding buffer. Then, 5 µL of Annexin V-APC and 10 µL of 7-AAD (MultiSciences Biotech, China) were added to the cells, which were then incubated in the dark at 25 °C for 25 min. The cells were immediately analyzed by flow cytometry.
To assess the effect of gene expression on cell apoptosis, cells were centrifuged and resuspended in 50 µL 1× binding buffer. Then, 5 µL of Annexin V-APC and 10 µL of 7-AAD (MultiSciences Biotech, China) were added to the cells, which were then incubated in the dark at 25 °C for 25 min. The cells were immediately analyzed by flow cytometry.
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