Cellular senescenceIt is a phenomenon in which cells become less functional and gradually die under normal environmental conditions.
DetectionCell senescence method steps:
1. Before cell seeding, pre-place sterilized cell pieces in 6-well culture plates, add 1 10e5 cells per well, and culture overnight under 37 5% CO2 conditions to make the cells grow on the cell sheets.
2. Aspirate the culture medium in the 6-well culture plate in the ultra-clean bench, add 1 PBS, wash the cells once, and then add 2% formaldehyde 02% glutaraldehyde fixative solution, fix at room temperature for 3 5 min.
3. Absorb and discard 2% formaldehyde 02% glutaraldehyde fixative solution, add 1 PBS, and wash the cells 3 times for 3 min each.
4. Aspirate 1 PBS, add X-gal solution to submerge the cell slices, 37 incubate for 4 8 hours or overnight, and coat the 6-well culture plate with plastic wrap to prevent the dye from evaporating.
5. Take out the cell crawler, rinse with deionized water twice, fix with fixative solution again for 4 minutes, and gently rinse with running water.
6. Dehydrate the cell slides in this order, transparent 95% alcohol I (2 min) 95% alcohol (2 min) 100% alcohol i (2 min) 100% alcohol i (5 min) xylene i (2 min) xylene (2 min).
7. Neutral gum mounting.
8. Observe the morphology of senescent cells under an ordinary light microscope.
Count 400 cells under each microscope to determine the percentage of cells that are positive for X-gal staining (senescent cells) in the cell population, i.e., the senescent cell rate (number of blue-stained cells, total number of cells, 100%).
Precautions:
X-gal solution needs to be prepared ready-to-use.
Cells that are fixed for too long or are not cleaned after fixation will affect the subsequent enzymatic reactions.
PCR test kits