WangLab:Passing Cells

Description
When cells are confluent, we pass them from one dish to three dishes, to synchronize the cell growth cycle and prepare for experiment.

Materials

 * 1) PBS
 * 2) trypsin, 1x for smooth muscle cells and 0.5x for endothelial cells (warm up in water bath)
 * 3) DMEM (with calcium, warm)
 * 4) DMEM with 10% FBS (warm)
 * 5) 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).

Procedures

 * 1) Rinse confluent cells with PBS for 3 times
 * 2) 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.
 * 3) Quickly add DMEM (with calcium) to neutralize trypsin.
 * 4) Pipet some medium to blow cells into suspension. Double check under microscopy to make sure all the cells are in suspension.
 * 5) Collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
 * 6) During the centrifuging period, take 3 new tissue cultured dishes. Label the dishes with cell name, passage, date, initials of your name.
 * 7) 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.
 * 8) Swirl the dish gently to allow the cells to spread evenly throughout the dish.
 * 9) Keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.