Hello,
I am trying to perform a respiratory syncytial virus (RSV) plaque assay. I can see the syncytial formation, white dots with a crystal violet background (3 replicates). However, I don't see consistency. What could be the reason? I am using Vero cells, confluence 90%, infected, and incubated with dilution for 1 hour with frequent rocking every 20 minutes. Overlayed with 0.6% methylcellulose in media (at 37C). Incubate for 5 days. Remove overlay and stain with crystal violet for 30 mins. Washed with running water.
Pictures for reference.
Thank You.
Possable getting secondary plaques on the mono layer which may not help. CMC overlay will only partly stop virus spread. Caution to not disturb the mono layer during incubation ie don’t remove plates during incubation to view on the microscope. Try and reduce the incubation time a bit as this will help prevent secondary plaques formation. Check/count on the microscope to confirm that each is truly a viral plaque based on syncytial type morphology.
Even after optimising the assay, it’s best to compare test samples from one experiment in the same assay ie all together.This prevents the problem of any variation from the assay itself, on different days, confusing things.
