Quick Guide to Plaque Assays

Last week we ran a short training course on baculovirus expression vectors.  As part of this we put together a one page guide for carrying out plaque assays.  This might be useful to those of you looking for something you can have on the lab bench while conducting your own plaque assays.  We have added a image of one of our 12-well plate-based plaque assays below as an example.  The plate was stained with neutral red four days’ after the assay was set up.
If you don’t fancy doing your own plaque assays on virus stocks we can do for them you as part of our services for customers (for a reasonable charge of course!).

Plaque Assay Protocol for Recombinant Viruses

This protocol is intended as a short description for use in the lab with 12-well plates.  A full method is in the baculoCOMPLETE manual.

1. Set up the required number of 12-well plates with 0.5 x 10⁶ Sf21 cells/well/1ml TC100/10% FBS. Incubate for 1-2 h at 28°C before use.  Alternatively, use 0.4 x 10⁶ cells/well/1ml TC100/10% FBS and incubate overnight.  One plate per virus sample is convenient.
2. Dilute virus stocks in TC100/10%FBS using ten-fold steps (e.g. 10^-1 to 10^-6). For multiple samples it is convenient to use a 48-well plate.
3. Remove medium from the cells in each 12-well plate just prior to adding the virus dilutions (treat one plate at a time).
4. Inoculate cells in the centre of the well with 100µl virus dilutions according to your requirements (e.g. 2 wells/dilution for 10^-1 to 10^-6; 3 wells/dilution for 10^-3 to 10^-6; 4 wells/dilution for 10^-1 to 10^-6).
5. Incubate plate at ambient temperature for one hour on a rocking platform if you have one, or manually rock plate from side to side every 10-15 minutes.
6. During the one hour incubation period prepare overlay medium. Start by warming TC100/10% FBS to about 37°C.  About 15 mins before you need to overlay your plates, melt a stock of 2% low melting temperature agarose (sterilized) using a microwave.  Just before the one hour is finished, mix equal volumes of TC100/10% FBS with hand hot 2% agarose.  Prepare at least 10% more than you actually need to allow for pipetting errors. Place mixture in water at 37°-42°C to prevent setting.
7. When one hour incubation is finished, remove virus from one plate at a time using a 1ml pipette (e.g. Gilson P1000).
8. Immediately, add 1ml agarose/TC100 overlay medium to each well. Remember to keep the mixture warm while you are dispensing it.
9. Repeat for all plates you are working with.
10. Allow overlay to set – takes about 15min depending on your ambient temperature.
11. Add 1ml TC100/10% FBS to each well.
12. Incubate for 4 days at 28°C.
13. At day 4, stain plaque assay with neutral red.