Separating and Passaging Cells in Tissue Culture

Splitting and Passaging Cells in Tissue Culture

1. Media warm and Trypsin-EDTA in a water bath (Trypsin-EDTA are made by diluting the stock by adding PBS 1/10 only)
1. cell divisions when theyre about 80-90% confluent
2. wash the cells with ~ 7ml PBS-EDTA (1 mM EDTA)
3. The aspiration of PBS-EDTA
4. + 1mL of trypsin per 100mm plate
5. incubate at 37 degrees C incubator for 1-5 minutes (usually 1 min.); Dont do it longer because it will damage the cells.

Separating and Passaging Cells in Tissue Culture

6. took the dish from the incubator and bang the side dishes around
7. Take a long pipette and pipette the solution up and down to break up any clumps of cells (if it’s really important to get them broken to count cells for cloning or limiting dilution to a single clone out, you might see under a microscope to see whether all clumps of cells have been damaged)
8. point solutions into a sterile plastic tube with FBS-containing media in it (FBS inactive trypsin, which is why it should be rinsed with PBS-EDTA initially) a final concentration of ~ 1% FBS should be enough to turn off trypsin
9. use the tube to balance to balance the tubes with cells, spinning for a few minutes (~ 2 minutes should be sufficient)
10. aspiration off media
11. resuspend the desired amount of media (if suspend media 10 ml, cells of only the first resuspend in 1 mL, then resuspend at rest, because it is easier to break up the clot using a small volume of media)
12. dilute the appropriate amount (usually around 1/10 for a few days) in the new dish containing the new media