Hybridoma culture problem. Help! - (Jan/03/2011 )
Dear forum members,
I am experiencing big problems lately while culturing my hybridomas. Cells are ok at the beginning but then, they start to vaculize and finally they die.
This is happening with already stablished hybridomas and also after fusion (cells start to form colonies but then, they die).
This is a recent problem (less than 1 month old) and we are using the same batches of RPMI,serum,antibiotics and plasticware as before. Moreover, we tested mycoplasma contamination and the results were negative. No yeast or bacteria contamination has been detected. At present, we are checking the incubators...temperature is ok but CO2 level has to be tested yet. However, since we have four incubators and the problem is present in cells incubated in all of them, I think the problem has to be somewhere else.
Any ideas?
Thanks a lot in advance!
Chelo on Mon Jan 3 12:01:22 2011 said:
Dear forum members,
I am experiencing big problems lately while culturing my hybridomas. Cells are ok at the beginning but then, they start to vaculize and finally they die.
This is happening with already stablished hybridomas and also after fusion (cells start to form colonies but then, they die).
This is a recent problem (less than 1 month old) and we are using the same batches of RPMI,serum,antibiotics and plasticware as before. Moreover, we tested mycoplasma contamination and the results were negative. No yeast or bacteria contamination has been detected. At present, we are checking the incubators...temperature is ok but CO2 level has to be tested yet. However, since we have four incubators and the problem is present in cells incubated in all of them, I think the problem has to be somewhere else.
Any ideas?
Thanks a lot in advance!
Have you tried the HAT medium? You should use enhancing medium or LPS/Dextran Sulphate
Some colonies may be unstable SO they die.
Have you checked for viral contaminants?