Tissue culturing - Crash down of culture -Unknown reason!! (Sep/21/2007 )
Hi
can someone please provide some helpful opinion here.
The tissue culture at my place is grown ridden with a strange problem.
Cells like HeLa are growing slow like snails and many of them aquire vauoles in the cytoplasm.
Other sensitive cells like C2C12 myoblasts are simply devastated showing an ELONGATED, GROWTH STAGNATED AND HEAVILY VACUOLATED CYTOPLASM staus.
The medium has to be changed too frequently and there are many cells that have an unhealthy look.
It seems the Medium is losing its buffering capacity although we haven't changed the preparation method or any composition.
What can be done??
The serum change (to a different batch) appeared to rescue my C2C12 for a while (P4)mbut they still seem to be a bit stressed with the acidic pH and get vauolated and elongated if left without serum change every 12-28 hrs.
where have we gone wrong??
Are you culturing the two lines at the same time in the hood? or using the same media bottle to culture the lines?
have you tried decontaminating the hood and incubator?
It's a pain but it has to be done.
Hope that helps.
1) Do you have L-glutamine in your medium?
2) Is mycoplasma infection a possibility?
have you tried decontaminating the hood and incubator?
It's a pain but it has to be done.
Hope that helps.
No, C2C12 cell lines are so fussy they need 20% FCS supplement so they are always handled separately.
DAPI never showed any sign of mycoplasma but occasionally we do have bacterial and fungal contamination. more frequent than earlier. i guess we are going to get the place fumigated.
thanks for the advice.
2) Is mycoplasma infection a possibility?
Yes it has, we also replenish it with 4mM L-glutamine later if the Incomplete is more than 10 days old.
I checked up the DAPI for mycoplasma and there is no cytoplasmic specks seen .
But is it possible that the pathogen is able to hit so drastically that the cell physiology is messed up without a visible trace upon DAPI staining?
The strangest part is that when i changed the medium FCS with some other lab's stock .....it helped.
I am spooked as to how come there is this relapse.