I am working with Rota virus and I am using MA104 cell line for propagation. I am not able to differentiate between mock flask and infected flask as it involves the addition of trypsin for both activation of virus and media used for the propagation. Does anyone have any more procedures related to the propagation of Rota virus and it's storage.
Hi; you should see a differnece between mock and rotavirus infected cells (if the virus has a sufficient titer... if not make several passages): MA104 resist very well to trypsin concentration (1ug/ml) used for rota propagation.
before infection don't forget to wash (3 times) your cells with PBS or medium without serum to remove trypsin inhibitors present in the serum. and then propagate the virus in a serum-free medium with trypsin.
After an infection you should get a "flag" lysis= the cells slide from the flask when you move it.... starting with a low (0.01) moi you should get complete cytopathic effect (no more cells attached to the flask) in 24-48 hours post infection.
then freeze thaw the culture 3 times, centrifuge 10K 20 min to remove cells debris and store the virus aliquoted below 70°C. titrer the virus (plaques or TCID 50), never re-used thawed virus. for short term storage 4°C is OK.
for human on non-adaptated rotavirus the cytopatic effect can be less pronounced but cells will detached "individually' and you get a cloudy culture medium. Still you should have a clear differnece between infected and infected cells ....
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