Pi rnase staining solution

Tumor cells were treated with 10μM cisplatin or paclitaxel for 12 hours, respectively, followed by 2% hexanediol together with the chemotherapeutic drugs for another 12 hours. Then, about 5×10⁶ cells were collected and washed with ice-cold phosphate-buffered saline (PBS) and fixed in 5 mL 70% ice-cold ethanol overnight at 4°C. Subsequently, the cells were pelleted and stained with 500 μL PI/RNase staining solution (KeyGEN Bio TECH, KGA512) in dark for 60 minutes followed by 3 washes with PBS. Fluorescence of PI was measured at an excitation wavelength of 488 nm by flow cytometry on a MoFlo XDP flow cytometry (Beckman, USA) and about 20,000 cells were collected for analysis.

With the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (KeyGEN BioTECH, China), apoptosis was detected. A total of 1.2 × 10⁶ cells were seeded in 10 cm cell culture dishes. A 12-h incubation with DMSO (Solarbio, China) or PEITC (Sigma-Aldrich, Missouri, United States) was carried out after overnight incubation. Following harvest and washing with PBS, the cells were suspended in 500 µL binding buffer containing 5 µL of annexin V-FITC and 5 µL of PI for 15 min at room temperature out of the light. Apoptosis was analyzed by an ACEA NOVOCYte3130 flow cytometer (Agilent Technologies, California, United States).
For cell cycle analysis, a total of 1.2 × 10⁶ cells were seeded in 10 cm cell culture dishes. A 12-h incubation with DMSO or PEITC was carried out after overnight incubation. After harvesting, for fixing, the cells were rinsed with sterile PBS, then centrifuged (2000rmp for 5min, 4°C), washed twice, counted, and resuspended in 70% cold ethanol for 2 h at −20°C. After that, the cells were washed and centrifuged twice, resuspended in 500 µL of PI/RNase staining solution (KeyGEN BioTECH, China) and incubated in darkness for 30 min. The samples were analyzed using an ACEA NOVOCYte3130 flow cytometer (Agilent Technologies, California, United States).

HaCaT cells were divided into control, UVB, UVB + EGCG (50 μM), and UVB + CBD (4, 8, and 16 μM) groups. After the UVB irradiation or sham irradiation, the cells were incubated with different treatments for 24 h. The cells were then collected, fixed, and permeabilized using 70% ethanol overnight at 4 °C. Subsequently, the cells were intracellularly stained with 2 μL of H2A.X Phospho (Ser139) FITC (BioLegend Technology, San Diego, CA, USA) for 1 h and then stained with 500 μL of PI/RNase staining solution (KeyGen Biotech, Nanjing, China) for 30 min. Finally, the cells were detected by flow cytometry.