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Help-Plaque assay-HSV infected Vero E6 - (Apr/18/2011 )

Hi everyone. I am doing a plaque assay of using HSV virus to infect Vero E6 cell line.

1) 6 well plate with virus dilution from 10^-2 to 10^-7
2) Dilute the virus with 1X MEM (1% FBS)
3) Wash cells twice with 1X MEM (no FBS)
4) After 1 hour infection in an incubator, placing low melting temperature agarose on top ( 2X MEM, 2% FBS + LMT agarose)
5) After 72 hours infection, Fix and stain the cells

Basically, I can observe plaques in the dilution of 10^2- 10^5. However, when I remove the agarose layer, the cells come of with the overlay. Therefore, I cannot fix and stain the cells. Should I fix the cells first then remove the overlay or anyone can suggest me a better idea? Currently my lab is using 7.4%Formaldehyde/PBS for fixing and 0.1% crystal violet for staining.

I will be glad if anyone can help. Thanks


You aren't supposed to remove the agarose before fixing and staining. Add your fixative straight on top of the agarose and then incubate at room temperature (overnight should do it). Then you wash the agarose off before reading your plaque assays.