2.6 Flow Cytometry for Determining Cell Apoptosis Rate and Cell Cycle PI Staining Method

2.6 Flow Cytometry for Determining Cell Apoptosis Rate and Cell Cycle (PI Staining Method)

Under sterile conditions, take 0.5cm of fresh tumor tissue, place it in a petri dish, use phosphate-buffered saline (PBS) to remove blood and clots from the surface of the tumor tissue, add a small amount of PBS; use ophthalmic scissors to cut the tissue into a homogenous slurry, add 5ml PBS; use a pipette to draw up the tissue slurry, filter it through a 200-mesh nylon sieve into a test tube; centrifuge at 1000r/min for 5 minutes. Then wash three times with PBS, each time centrifuging at a low speed of 800r/min for 5 minutes to remove cell debris; filter through a 350-mesh nylon net to remove cell clumps, collect 200ul of single-cell suspension, add 0.5ml of 75% ethanol, fix at 4℃ for 12 hours, wash away the fixing solution with PBS two to three times, centrifuge at 1000r/min for 10 minutes each time, discard the supernatant and keep the sediment. If the cell count is above 1×10⁶ cells/ml, take 50ul of the suspension, add 15ul of pyridine iodide, stain for 30 minutes. Analyze using a flow cytometer.