Hi, I want a correct protocol for plaque assay with influenza viruses .
I used MDCK CELLS ( 450 000 cell/well ) with different strains and different plates but i didn't see plaque formation but only holes on all conditions ( also control).
What do you think about this problem?
Can i have a correct protocol of it?
thank you
best regards
When plaquing IAV on MDCKs I use 12-well plates. 300'000 cells/well seeded the day before. This keep them confluent in a monolayer but not so dense that your plaques will end up 'fuzzy'at the end.
Day of infection: 1) remove media and wash with PBS. 2) Remove wash and infect using a 100uL inoculums of serially-diluted IAV suspended in virus growth media. 3) Incubate for 1 hour, rocking every 15 minutes so your cells don't dry out. 4) Add 1 mL per well of your overlay. I use an overlay consisting of equal parts 2xDMEM and 2.4% avicel (so 1.2% final conc). This will contain TPCK-trypsin depending on the type of IAV I'm plaquing.
Next, let the plates incubate for 2-3 days.
To finish, aspirate your overlays and wash with PBS. Fix the cells for 10 minutes using 70% ethanol. Remove the ethanol and stain the wells using 0.3% crystal violet for ~10 mins.This will allow easy visualization of plaques.
Things that can be optimized depending on the influenza virus you're using & how well your cells grow, etc:
- cell seeding density, composition of virus growth media, presence/absence of TPCK-trypsin, how many days you let plaques form post-infection.
Camilla, from your protocol that you sent, you did not add TPCK-trypsin, which is required for your virus in order to visualize plaques. You should use 1 - 2 ug/ml TPCK-trypsin to your cultures. This should allow you to see nice plaques withing 2-3 days.
Thank Thomas fot your advice . I try it and then i’ll contact you . Thanks so much for your precious answer.
@ Cason King
how do you make 2.4% Avicel solution. I can't dissolve them in water.
thank you
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating