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. setrile 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 2 ml trypsin, keep in 37oC for 1.5 min, not to over 2 min.
3. Gently pat the dish, check under microscopy to make sure all the cells are in suspension.
4. If the cells are not in suspension, pipet up and down several times to blow the cells into suspension.
5. Quickly add DMEM (with calcium), 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 10% FBS DMEM by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled dishes.
8. Shake 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.

Notes

• The amount of medium is decided by the size of the cell culture dishes.
• If you are passing cells for transfection, use DMEM without antibiotics.
• The optimal time interval between passing cells and transfection is 15-18 hours; and that between transfection adn imaging is 36-54 hours.