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293T Cell Culture Protocol

Buffers:
Phosphate-buffered saline (PBS)

Adjust pH to 7.2-7.4. Autoclave before use.

Cell culture: 293T cells were grown in DMEM (invitrogen) supplemented with 10% FBS (Hyclone) at 37oC in 5% CO2 using 100mm culture dish.

Subculture:

1. Check the confluence of the cell using an inverted microscope. Prepare to sub-culture the cells when they reach ~80% confluence.
2. Carefully aspirate old medium from the cells.
3. Gently wash the cells with 3ml PBS.
4. Add 3mL of new media to resuspend cells by pipetting up and down, and avoid frothing of medium.
5. Pipette 700ul of the cell suspension into a new 100mm culture dish containing 10-12mL new media.
6. Renew the media after 24h.
7. Cells should be reaching 80% confluence again after 48h of last subculture.