Cytoflex srt flow cytometer

The initial stages of sample preparation were analogous to the above. After washing the cells twice with PBS, the cells were centrifuged at 1000 rpm for 10 min and then the supernatant was aspirated. The cells were vortexed (to loosen the pellet) by dropwise addition of 5 mL of cold 70% ethanol and incubated at −20 °C overnight. After this time, the cells were washed twice to remove the ethanol. The cells were then centrifuged for 10 min at 1000 rpm and the supernatant was aspirated. For cell staining, 0.5 mL of PI/RNase staining buffer (BD Pharmingen, San Diego, CA, USA) containing propidium iodide and RNase was added to each sample. The cells were incubated with the dye for 15 min at RT. Samples were stored at 4 °C and protected from light until analysis. Stained cells were analyzed on a CytoFlex Srt flow cytometer to determine the relative DNA content, which was based on red fluorescence intensity.

Cells were seeded into cell culture flasks (T75) (5 × 10⁵ cells). On the second day, the medium was changed, and the cells were treated with a fisetin derivative. Cells were incubated for 24 h before harvesting. The cells were washed with a wash buffer and gently fixed by adding cold 75% ethanol (cyclin B1) or 75% methanol (cyclin D1) before being placed in a freezer for min. 2 h. Next, ethanol/methanol was removed, and the cells were treated with 0.25% Triton X-100 for 5 min in an ice bath. The cells were washed and resuspended in a wash buffer at a final concentration of 1 × 10⁷ cells/mL. Then, 100 μL of obtained cell suspension was transferred to new tubes and stained by adding 20 μL of FITC cyclin B1 or FITC cyclin D1 and incubated in the dark for 30 min at RT. After 30 min of incubation, the cells were washed and resuspended in 0.5 mL 7-AAD solution and incubated for 20 min at 4 °C. Then, the cells were analyzed using a CytoFlex Srt flow cytometer. For each measurement, at least 10,000 cells were counted.

Cells were seeded into culture dishes at a density of 5 × 10⁵ cells/dish and incubated for 24 h at 37 °C and 5% CO₂. After incubation, cells were stripped and then washed twice with PBS. The apoptotic effects of the flavonols were analyzed using the Annexin V Apoptosis Detection Kit II (BD Pharmingen, San Diego, CA, USA), which contains Annexin V binding buffer -FITC and PI staining buffer.
Briefly, cells were suspended in 100 μL of binding buffer and sequentially mixed with 5 μL of Annexin V-FITC and 5 μL of PI. The mixture was incubated for 15 min at room temperature in the dark. Cell apoptosis was quantified using a CytoFlex Srt flow cytometer (Beckman Coulter, Miami, FL, USA) and dedicated CytExpert Srt software Version 1.1.