Dav 1 (or when confluents 
1. Aspirate off media from cells with a pasteur pipette. 
2. Use a 5 ml pipette to add 5 ml PBS to each plate. 
3. Aspirate off PBS with a pasteur pipette. 
4. Add 1 ml of Trypsin-EDTA with a 1 ml pipette to each plate. 
5. Let plate sit in hood until cells just start to release from the plate. 
6. Add 5 ml of complete medium to each plate with a 5 ml pipette (to stop the trypsin 
reaction) and transfer to a 15 ml conical test tube. 
7. Spin down each test tube in a clinical centrifuge at setting 3 for 2 minutes. 
8. Aspirate off the supernatant with a pasteur pipette. 
9. Resuspend each cell pellet with 10 ml of complete medium using a 10 ml pipette. 
10. Add 1 ml of cell suspension to a 10 cm plate (that had already been filled with 9 ml 
of complete medium while the cells were being spun down). This constitutes a 1:10 
split. 
11. Place each plate (50 total) at 37 °C, 5% CO2. 
Dav 5 (or when confluent 
1 . Aspirate off media from cells with a pasteur pipette. 
2. Use a 5 ml pipette to add 5 ml PBS to each plate. 
3. Aspirate off PBS with a pasteur pipette. 
4. Add 1 ml of Trypsin-EDTA with a 1 ml pipette to each plate. 
5. Let plate sit in hood until cells just start to release from the plate. 
6. Add 5 ml of complete medium to each plate with a 5 ml pipette (to stop the trypsin 
reaction) and transfer to a 15 ml conical test tube. 
7. Spin down each test tube in a clinical centrifuge at setting 3 for 2 minutes. 
8. Aspirate off the supernatant with a pasteur pipette. 
9. Resuspend each cell pellet with 10 ml of complete medium using a 10 ml pipette. 
10. Add 1 ml of cell suspension to a 10 cm plate (that had already been filled with 9 ml 
of complete medium while the cells were being spun down). This constitutes a 1:10 
split. 
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Recombinant DNA Research, Volume 15 
