2.7 Cell Apoptosis Rate and Cell Cycle Determination

2.7 Cell Apoptosis Rate and Cell Cycle Determination

Under sterile conditions, take 0.5 cm³ of fresh tumor tissue and place it in a petri dish. Use phosphate-buffered saline (PBS) to remove blood and clots from the surface of the tumor tissue, then add a small amount of PBS. Use ophthalmic scissors to cut the tissue into a homogenized state, and add 5 ml of PBS. Use a pipette to aspirate the tissue homogenate and filter it through a 200-mesh nylon mesh into a test tube. Centrifuge at 1000 r/min for 5 minutes to precipitate the cells. Wash three times with PBS, each time centrifuging at a low speed of 800 r/min for 5 minutes to remove cell debris; then filter through a 350-mesh nylon mesh to remove cell clumps. Take 200 μl of the single-cell suspension, add 0.5 ml of 75% ethanol, and fix at 4°C for 12 hours. Wash away the fixative solution with PBS 2–3 times, centrifuging at 1000 r/min for 10 minutes each time, discarding the supernatant and retaining the pellet. If the cell count is above 1 × 10⁶ cells/ml, add 50 μl of the suspension to 1000 μl of propidium iodide and stain for 30 minutes. Prepare for instrument-based detection [22–24].