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I got stuck TCID50 test in my PR/8 virus strain in MDCK cells.
My MDCK cells are 4+ split after getting from ATCC company (CCL-34) and PR/8 strain were passed 8th in Eggs.
I actually did TCID50 last year and it work fine. The titer is like 5logTCID50
But recently I couldn't get the same titer and it's like the cell lost cytopathic pattern after 10^-2 virus dilution.
Here's my protocol:
1. Grow MDCK cells in T-75 flask .
2. Split cells and spread them into 96 well plate after 48h incubation.
3. Grow cells up in 96 well plate for 24h until it gets 90%> confluency.
4. Add 180ul 5%FBS containing DMEM media into another 96 well plate.
5. Add 20ul virus per well to the first column. Transfer 20ul from the first column to the 11th to make virus dilution from 10^-1 to 10^-11 in the 96 well plate and leave the last column for control.
6. Remove the media from 96 well plate and add 100ul virus dilution to each well.
7. Put the plate into 37oC incubator for 2h.
8. remove the media from the 96 well plate.
9. Wash the 96 well plate with 100ul 5%FBS DMEM media,
10. Add 200 ul 5%FBS contained EMEM (ATCC suggested) to each well to continue incubation
11. Fix cells after 4 days' incubation by using 100ul 1% Formaldehyde.
12. Stain cells with 0.1% crystal violet for 1h.
I only got 10-1~10-2 TCID50 now.
It is wiered cause I got 50% cell death after 2 days in T-75 flask when I add 10^-1.5 dilution in the flask. But I got little death in 96 well plate after 4 days' incubation
Also, the media in 96 well plate changes color quickly, it turns to orange and even faded. I think that's because that PH has changed/
Anyone can help me on this problem?
Thanks a lot!
