I was performed TCID50 to test influenza A PR8 for several times. But the MDCK cell cannot survive in the negative control after the medium was changed from DMEM medium to TPCK-infection medium.

I suspect there is some problem with my TPCK-infection medium.

The experimental process and medium recipe are as follow:

1.Seed MDCK cells in 96 well plate at 1.5*10^4 cells/well in DMEM (200ul total) for about 1 days in 37°C,5%CO2.

2.On the day of infection, remove the DMEM and wash the cells twice with PBS.

3.Add 100 μl 1:10 serial diluted virus with TPCK-infection medium in 37°C,5%CO2 for 2hr.

4.Remove the medium and wash the cells once with 150ul PBS.

5.Add 200ul TPCK-infection medium and Incubate the plates at 37°C,5%CO2 in incubator for 96 hr.

5.After 96 hr ,remove the TPCK-infection medium and add 100ul 4% formalin for 10 mins in room temperature to fix the cells.

6.Remove the formalin and stain the cell with 1% crystal violet for 10 mins in room temperature.

7.Wash out the crystal violet and give each well positive or negative score.

DMEM medium:

DMEM/high glucose(HyClone)

10% FBS

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TPCK-infection medium:

DMEM/high glucose(HyClone)

0.75% BSA

25mM HEPES

2ug/ml TPCK-trypsin

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