When cells are confluent, we pass them from one dish to three dishes, to synchronize the cell growth cycle and prepare for experiment.
- trypsin, 1x for smooth muscle cells and 0.5x for endothelial cells (warm up in water bath)
- DMEM (with calcium, warm)
- DMEM with 10% FBS (warm)
- sterile cell culture dishes (if not tissue culture treated, coat the dishes with 2 % gelatin (just rinse), if not sterile, incubate with ethanol or light-bath with UV lamp for 30 min and then rinse with PBS for 3 times).
- Rinse confluent cells with PBS for 3 times
- Incubate cells with 0.5x trypsin (1 ml for medium dish and 2 ml for large dish), keep in 37oC for 1.5 min, not to over 2 min.
- Quickly add DMEM (with calcium) to neutralize trypsin.
- Pipet some medium to blow cells into suspension. Double check under microscopy to make sure all the cells are in suspension.
- Collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
- During the centrifuging period, take 3 new tissue cultured dishes. Label the dishes with cell name, passage, date, initials of your name.
- Take out the centrifuged tube containing cells, you should be able to see a whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant with vacuum tip, resuspend the cell pellet with 3 ml 10% FBS DMEM by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled dishes, add more 10% FBS DMEM according to the dish size.
- Swirl the dish gently to allow the cells to spread evenly throughout the dish.
- Keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.
- The amount of medium can be decided by the size of the cell culture dishes.
- If you are passing cells for transfection, use DMEM without antibiotics.