Few weeks back there was fungal infection in some of my petri dishes. So, I thoroughly cleaned the incubator using bleach and rectified alcohol, revived fresh stocks, prepared everything fresh and started using sodium azide in the humidity pan 1g/L autoclaved water (earlier I used to use copper sulphate 1g/L). Now, the infection has disappeared but my cells are dettaching after the first splitting I do after stock revival. what could be the reason for it.
Have you checked viability of cells after trypsinization/thaw? Have you changed serum lot in between? Can you grow any other cell types (adhered) in the same medium and serum?
I agree with comments from James and Asok. Further, I would like to add that a number of factors could contribute to the lack of attachment of your cells. Therefore, you need a systematic approach to rule out a number of possibilities starting with contamination of cells, defects in cell plates/flasks, and poor culture conditions involving serum etc. You may want to include a different cell line as suggested by Asok to rule out poor culture conditions.
it happens sometimes, you just check your media pH, and centrifuge your cells while doing trypsinization and add fbs 20% and 10 ml media in T-25 cell culture flask.
@James Joseph
There is no mycoplasma contamination I am sure about that. Yes I was getting a good no. of cells after revival but since yesterday even the stocks are not getting revived. I feel there is some problem with the incubator, maybe due to Sodium Azide or any other reason which I cannot decipher.
@Asok Mukhopadhyay
No sir, I have'nt checked the viability of the cells after trysinization and thawing. The serum lot is 5 months old, cells were growing properly in this lot. The response is same for all the cell lines of this and other media. I seriously cannot figure out what the problem is?
@Divaker Choubey
Sir, I have already tried that. I even took the stocks from other labs too.
azide in the humidity plan? i would never do this. one problem is the amount is lot to be dangerous. next, azide is some extent volitale at acidic pH only of course, but a low amount will evaporate in your incubator surely, might cause the cellular death or stress even. carefully change it back to Cu salt, please.
I agree with Attila; even a small amount of azide in incubator atmosphere can stress your cells. Or perhaps you have chlorine from bleach still blowing off from cleaned incubator parts.
Possible contamination, problem with medium, cell culture flasks, over trypsinization etc. Run a viability test. Leave the cells for a longer period. Check also incubation conditions. Good Luck
@Attila Lehotzky
I have removed everything from the humidity pan yesterday itself, now it is just autoclaved distilled water.
I am going to leave it for two days. Then again on Monday i will replace humidity pan water with some fresh autoclaved distilled water and then i will revive fresh stocks.
Hope it works..!!
@Anne T Quets
I cleaned it with bleach a month ago, will the chlorine blow still?
@Bichitra N Nayak
Sir, but I have checked for contamination, the cultures are infection free. How to run a viability test?
Himanshi
Let's assume your incubator is clean. I am unfamiliar with your cell line. If you are using a bicarbonate pH buffering system, it is critical that the CO2 in the incubator "matches" your growth media. BNN's suggestions are correct. The best international journals require cell line confirmation/documentation. Reacquire your cells from ATCC or an equivalent scource.
Michael
Hi Himanshi,
Have you changed to a different type of culture flask? Different brands are treated slightly differently and for some cells this can affect their adherence.
Good luck,
Kirsty
Sodium azide can react explosively with copper so beware!
Can you you measure CO2 in the incubator? (In the US the brand name chemical method is called Fyrite)
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