I am testing TCID50 of Influenza virus work seed batch with MDCK cell lines.The experimental process is as follows：
1.Put MDCK cells in 96 well plates at 16000cells/well in DMEM +10% FBS (100ul total) for about 1 days.
2.On the day of inoculation, remove the DMEM and wash the cells twice with PBS.
3.Add 200 μl 1:10 serial diluted virus with DMEM+3ug/ml TPCK- trypsin.
4.Incubate the plates at 34°C in a tissue culture incubator for 72 h.
5.Calculate the virus titer by determining the end point dilution that test positive for hemagglutination of RBCs.
There are currently some issues with this experiment, after 24 hours of step3, the cells all shrink. Who can tell me what happened, thank you.
The problem may be related to the amount of TPCK-trypsin used in step3 for MDCK cells. The concentration could be too high for the virus inoculation to work properly. A possible solution is to perform a titration experiment to find the optimal concentration of TPCK-trypsin for this procedure.
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