Seeding Passaging Freezing Cells

Seeding, Passaging and Freezing Cells

Seeding, Passaging and Freezing Cells

Seeding Cells

1. Cells: freeze slowly, thaw really FAST!!

2. 14 mL RPMI into C-75 flask

3. pour 1 mL of frozen stock cells

4. spread out cells by rocking flask

5. DMSO is toxic – freezing medium has 10% DMSO and 20% PBS

6. after seeding, take cells into 15 mL Falcon tube, spin down at 1600 rpm, get a pellet

7. pour our supernatant, and add 15 mL more medium

8. resuspend pellet in 3 mL fresh media, spin again, take out medium again

9. fresh 3 mL RPMI, resuspend, put into flask which contains 12 mL of RPMI

Passaging Cells

1. remove medium

2. 10 mL of PBS washes C-75 flask

3. remove PBS, put in 1.5 mL trypsin

4. spread trypsin around by rocking, incubate at 37 degrees for 10 minutes

5. take 3 new flasks, put in 12 mL medium into each (subculture depends on the cell line)

6. stop trypsinization by adding 5 mL of medium

7. distribute evenly the cells into the new flasks (2 mL per flask if 3 flasks)

8. after splitting, always need to alter/change the medium the next day

Freezing Cells

1. trypsinize cells as previously mentioned

2. put cells into 15 mL Falcon tube

3. spin down cells at 1200 rpm

4. suck out supernatant

5. resuspend in 1 mL freezing medium

6. transfer 1 into 2 mL cryo tubes

7. place in close container with dry ice while working

8. when finished put into liquid nitrogen

Please refer to our other cell culture protocols.

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