Sean Lauber:Culturing Lewis Lung Carcinoma Cells

Thawing the culture

1. Thaw a vial of 3 million cells/ml in a 37°C water bath

2. Add 1 ml of PBS and add this 2 ml to 8 ml of PBS

3. Spin at 1000 rpm for 3 min to pellet

4. Discard the supernatant and resuspend the pellet in the residual PBS by tapping

5. Add 10 ml of warmed 10% FBS/DMEM and resuspend by pipetting

6. Take 1 ml of this and put it into a T75 flask containing 14 ml of warmed 10% FBS/DMEM

7. After 2 days, the cells need to be split

Splitting the culture

1. Remove the media from the T75 and place it in a 50 ml falcon tube

2. Wash the plate with 5 ml of PBS and then remove this and place it in the falcon tube

3. Add 1 ml of prepared trypsin to the plate

4. Tilt the plate back and forth over a period of 3 minutes to lift the adherent cells

5. Add 10 ml of warmed 10% FBS/DMEM to stop the trypsin

6. Remove the media to the 50 ml falcon tube

7. Spin the falcon tube at 1000 rpm for 3 min to pellet the cells

8. Discard the supernatant and resuspend the pellet by tapping in the residual liquid

9. Add 10 ml of warmed 10% FBS/DMEM and resuspend the cells by pipetting

10. Remove 1 ml of the cell suspension and add to 14 ml of warmed 10% FBS/DMEM in a T75 flask

Freezing the culture

These cells are a mixed adherent/suspension population and do not form confluent monolayers. The cells instead will begin to clump together and float in the suspension, or remain adherent to the plate. It's important when working with these cells to split them before they begin clumping. Once they clump together it is very difficult to generate a single cell population, therefore I always try to split them as soon as I begin to see clumps forming (usually 2 days). When the cells are frozen it is also important to remember this and try to ensure that single cells, and not clumps, are frozen down to maintain consistency between batches.