I was reviving endothelial cells. When I added the cells to the DMEM, the medium immediately changes colour to purple. It was like a magic! Then one day after that, the medium returns back to red colour. But now the cells have sadly died. What is happening? The DMEM that I used has 3.7g/L of sodium bicarbonate.
Nurul: Check the recipe of your DMEM to be sure that there is buffer (20 mM HEPES for example) which can sustain short period of pH fluctuations (too alkalinic in your case).
I use 2.4 g/ml sodium bicarbonate and 5% FCS. I think you need more FCS, like 10%. Pellet cells before adding DMEM and use high cell seeding density (50,000 living cells/mL or more). As soon as they are confluent (perhaps in 24 hs) and DMEM turns yellow duplicate the seeding area (amplification). If cells did'n reach confluency in two days, change the medium.
Dear Ke-Wei, there is no HEPES in the DMEM.
Dear Maria, I added 10% FBS, 1% Pen-Strep, 1% L-Glut. I was reviving a new frozen vial, so I did not know the cell concentration.
It looks that the cap was not closed properly on the vial, or the cell solution standed a little bit long time before freezing. What is the ratio of the frozen medium and the fresh one? Use more fresh, at least ten times or over... but likely the cells was alredy died.
Hi Adhwa,
It sounds as though your freezing medium is too alkaline. After recovery, you should pellet the cells at low speed (approx.1000rpm/10 mins) then resuspend them in the DMEM you are using for growth. You may need to repeat this more than once to remove the freezing medium. When incubating from frozen, always use vented capped flasks in a 5% CO2 incubator for at least the first 24 hours. If the medium is too alkaline the cells will not attach to the flask and will die.
Hope this helps,
Carol
Dear Attila - It was a vial from ATCC, 5% (v/v) DMSO.
Dear Dr Carol - Hello! Nice to hear from you. Anyway it is not my freezing medium. It is from ATCC. And I used a vented capped flask in a 5% CO2 incubator. Now my cells have died. Would it be possible if I ask for endothelial cells from you?
If it was purchased from ATCC, you can ask a new stock mightbe giving some reason. You can order live cells too, probably; it is not essential to get frozen one.
I agree. If these have come directly from ATCC (not frozen) and you cannot get them to grow, you should contact ATCC for advice.
Dear Atilla and Dr Carol - I have contacted ATCC, however because it is already outside the warranty period, I am not entitled for a replacement vial.
It really must have been the pH of the freezing medium - purple indicates above pH 8, and that was after mixing, so the frozen cells must have been well above pH 8 and (as Attila said) probably dead already. They must have de-gassed (lost CO2) while frozen.
By the way, standard DMEM has 44 mM bicarbonate as buffer (3.7 g/L) and is designed for use with 10% CO2 to reach pH 7.4. (Dulbecco always used 10% CO2). If you want to use DMEM with 5% CO2 and still get pH 7.4, do check that the bicarbonate is lower, say 25 mM as in MEM (2.1 g/L). Standard DMEM with 5% CO2 gives pH 7.7, too alkaline for optimal growth. (This is without HEPES which is also not a standard component. 45 mM bicarbonate is already a strong buffer.)
DC Bennett gives great advice. Also, remember that an old glutamine solution will have ammonia which is toxic to cells.
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