plaque assay polio virus - plaque assay polio virus (Aug/20/2010 )
I am doing a plaque assay with HeLa cells and infecting them with poliovirus. I dont get plaques?
Method i use:
1. I take a 80-90% confluent plate of HeLa cells. I do the assay in the 6 well plates from Nunc.
2. Dilute the virus in serum free DMEM-10-2 through 10-11.
3. Wash the plates with 1X serum free DMEM.
4. I infect the cells with virus 300 ul for 30-50 minutes at RT/rocking.
5. Overlay with 5ml(2XRPMI without phenol red) + 2.5 ml 1.5% Agarose +2.5 ml 1.5% Agar.
6. keep in the 37 degrees incubator, 5% CO2 for 48 hours
7. Fix with 10%(v/v)formaldehyde in 1XPBS for 30 minutes.
8. Stain-0.5%(w/v) crystal violet in 10% EtOH for 5 minutes. and wash with water.
It is very hard to get plaque with HeLa cells. I spent a year to get that but no success. So we tried Vero cells instead, and they work absolutely fine.
so your problem is not the method., it is the cells. Although many papers show plaque results with HeLa, I still think HeLa is not good cell line to do plaque assay.
Thanks! I will do a plaque assay on Monday with vero cells and let you know how it goes.