Sean Lauber:Culturing Lewis Lung Carcinoma Cells

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5. Add 10 ml of warmed 10% FBS/DMEM and resuspend by pipetting
5. Add 10 ml of warmed 10% FBS/DMEM and resuspend by pipetting
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6. Take 1 ml of this and put it into a T75 flask containing 14 ml of warmed 10% FBS/DMEM
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6. Take 1 ml of this and put it into a T75 flask containing 13 ml of warmed 10% FBS/DMEM
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7. After 2 days, the cells need to be split
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7. After 2 days (~48 hours, NO LONGER), the cells need to be split otherwise clumps will form (and you'll never get single cells again)
==Splitting the culture==
==Splitting the culture==
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1. Remove the media from the T75 and place it in a 50 ml falcon tube
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1. Remove the media from the T75 and discard
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2. Wash the plate with 5 ml of PBS and then remove this and place it in the falcon tube
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2. Wash the plate with 10 ml of PBS, discard
3. Add 1 ml of prepared trypsin to the plate
3. Add 1 ml of prepared trypsin to the plate
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4. Tilt the plate back and forth over a period of 3 minutes to lift the adherent cells
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4. Tilt the plate back and forth over a period of 3 minutes to lift the adherent cells, knocking the plate against your wrist a couple of times towards the end of the 3 minutes
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5. Add 10 ml of warmed 10% FBS/DMEM to stop the trypsin
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5. Add 9 ml of warmed 10% FBS/DMEM to stop the trypsin
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6. Remove the media to the 50 ml falcon tube
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6. Remove the media (10 ml) to a 15 ml falcon tube, pipette up and down several times
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7. Spin the falcon tube at 1000 rpm for 3 min to pellet the cells
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7. Add 1.5 ml of this suspension (so a 1.5:10 or 3:20 split ratio) to 13 ml of warmed 10% FBS/DMEM, rock the plate up and down, left and right to distribute. Check with a microscope the appropriate density.
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8. Discard the supernatant and resuspend the pellet by tapping in the residual liquid
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8. Split again in 2 days.
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+
-
9. Add 10 ml of warmed 10% FBS/DMEM and resuspend the cells by pipetting
+
-
 
+
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10. Remove 1 ml of the cell suspension and add to 14 ml of warmed 10% FBS/DMEM in a T75 flask
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==Freezing the culture==
==Freezing the culture==

Revision as of 17:27, 25 January 2013

Contents

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 13 ml of warmed 10% FBS/DMEM

7. After 2 days (~48 hours, NO LONGER), the cells need to be split otherwise clumps will form (and you'll never get single cells again)

Splitting the culture

1. Remove the media from the T75 and discard

2. Wash the plate with 10 ml of PBS, discard

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, knocking the plate against your wrist a couple of times towards the end of the 3 minutes

5. Add 9 ml of warmed 10% FBS/DMEM to stop the trypsin

6. Remove the media (10 ml) to a 15 ml falcon tube, pipette up and down several times

7. Add 1.5 ml of this suspension (so a 1.5:10 or 3:20 split ratio) to 13 ml of warmed 10% FBS/DMEM, rock the plate up and down, left and right to distribute. Check with a microscope the appropriate density.

8. Split again in 2 days.

Freezing the culture

1. Grow enough cells for 6 or so T150s

2. When the cells are ready (ie when you would normally split them - when they're still almost all single cell colonies rather than clumps), trypsinize them and follow the splitting procedure to the point where you have the cell pellet. It's easier to reserve 1 50 ml falcon tube for each T150.

3. Combine all pellets into a single falcon tube in 10 ml of 10% FBS/DMEM

4. Count the cells by sampling 10 ul of this solution (with trypan blue = 10 ul sample + 20 ul trypan blue + 70 ul PBS).

3. Dilute the cells to 3,000,000 cells/ml in 20% FBS/DMEM/5% DMSO (use the 10% FBS/DMEM and add more FBS, then add DMSO last and quickly mix to ensure you don't have patches of concentrated DMSO which would kill your cells)

4. Dispense 1 ml to labeled cryogen tubes (I put an L on the top of the tube, then on the side I write LLC, the approximate passage, and the date)

5. Put in a mr freezie and put at -80°C for one day before putting in liquid nitrogen


Notes

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.

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