plaque assay polio virus
Posted 20 August 2010 - 11:05 AM
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.
Posted 21 August 2010 - 06:02 AM
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.
Posted 21 August 2010 - 06:12 AM
Thanks! I will do a plaque assay on Monday with vero cells and let you know how it goes.