Passaging / splitting cells

Aim:

To passage / split a semi-confluent T25 or T75 cell culture flask to prevent cellular overgrowth or to expand cell cultures into additional or larger flasks.

Steps

1. Remove media

2. Wash 1 – 2 times with room temperature PBS. (2mL for a T25 flask or 6mL for a T75 flask).

3. Add 0.05% Trypsin & incubate for approximately 5 minutes at 37°C. (0.5mL Trypsin for a T25 or 1.5mL Trypsin for a T75).

Trypsin is a mixture of proteases widely used for cell dissociation.

4. Dilute Trypsin by adding media. (4mL for T25 or 6mL for T75 = >1:4 Trypsin:media).

5. Transfer to 15mL tube, centrifuge at 1000 rpm for 5 minutes & pour off media to remove trypsin. Resuspend gently in 5mL media.

6. If starting with a T25 and transferring to a new T25: Transfer 0.8mL for a 1/6 split and add 5mL media. Discard remainder. If starting with a T25 and transferring to a new T75: Transfer 2.5mL and add 10mL media. Discard remainder. (For fast growth transfer 5mL and add 7mL media). If starting with a T75 and transferring to a new T75: Transfer 0.8mL for a 1/6 split and add 11mL media. Discard remainder. If starting with a T75 and transferring to multiple T75 flasks: For 4 x T75 transfer 1.25mL per T75 and add 11mL media.

7. Incubate at 37°C for 24 hours then change media to remove any dead cells.