i'm working on MOLT-4 (acute lymphoblastic leukemia). It grows as suspension and I want to know more information about which is the best culture method to separate viable cells from dead cells.
You could try separating them using Ficoll.
http://www.upci.upmc.edu/cytometry/pdf/02FicolHypaq.pdf
http://www2.egr.uh.edu/~nvaradar/private/Prots/Fico_LD.pdf
Dear Fatemah Etezadi,
My suggestion would be to use gravity sedimentation procedure.
Shake the cell culture suspension and keep it for some time allow the cells to sediment by themselves.slowly remove the supernatant whcih will contain most of the dead cells and small density cells.Do it for 2 to 3 times Inshaallah you will be able to remove the dead cells from live cells.
Parveen
Ficol is a good idea. Also before that try separating the viable and dead cell by simple centrifugation. Try spinning the cells at 900 RPM for 5 mins and remove the supernatent. Repeat the process 3 times and let the culture rest over night and then check day after.
Centrifugation at 800 g for 5 min. Re-spinning of the supernatant at 1500 g for 5 min will allow you to see a thin pellet represented by dead cells and confirm the separation.
Dear Fatemeh
In my opinion, It would be useful to transfer your cell suspension into a 15 ml falcon and spin it at 1500 rpm for 10 min. Discard supernatant and add fresh medium to your pellet.
Good luck.
Please report centrifuge speed/gravity in g instead of rpm, this makes it universal for all types of centrifuges and independently of the used rotor diameter
For dead cell exclusion I would suggest 5 min 200g when purification is more important than cell recovery (mosty ~50%).
It is important to precise whether:
1) you want to exclude apoptotic/necrotic cells
2) you need it for general mentenance of cell culture or you want to purify cells just prior to an important experiment. In the second case you may also consider use of cell sorter.
There are 2 essential methods to maybe achieve what you want, centrifugation and single cell cloning.
Spinning the culture in a 15 ml Falcon tube at 300 x g for 10 minutes. Pour off the fluffy cells which are mostly debris, resuspend the cell pellet in fresh medium.
For single cell cloning, you will have to use a combination of dilutions and cloning dishes to get down to single cells and regrow. Both of these work well and are trusted classic methods.
You can also place your flask on a 45°angle.
Let gravity saparate te living cells to the bottom.
Then either remove the top layer of culturemedium.
Or suck the living cells from the bottom.
Done it a few times with very slow and messy growing cell lines.
We use low speed centrifugation (90g for 10 min) to eliminate debris and dead cells
it works well with cells that grow in suspension
You can start with 50g for 10 min and check your cells under the microscope. For me 25g for 10 min works pretty good.
Please spin down your cells in suspension at 500g for 10 mins. This keeps the dead cells in the supernatant. Resuspend your pelleted cells in fresh media. This method should remove most of the dead cells.
I heard the other way around but I am not sure 100%. Centrifuge at 800-1000xg for 3-5 minutes, so that nearly almost dead cells will burst and will be in the supernatant. might not be logical but if anyone can explain better, I would be appreciated. I always tried to use 300-400xg to remove dead cells before.
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