We want to count the titer of the baculovirus which does not include polyhedrin protein. We are not able to calculate them because they do not clearly form plaque in agarose. We tried to do that by staining the cells with neutral red. Do you know an alternative method?
Please inform me about it,
Hi Aydin,
the plaque assay for baculovirus can be quite tricky because many things can go wrong if you are not careful. I recommend you to let the agarose dry for at least 30 min before placing it in an incubator and waiting for 4-5 days before neutral red staining. I generally use the protocol from Berges lab (https://bergeslab.byu.edu/Portals/29/Protocols/Baculovirus%20methods.docx), maybe you would like to try this one out.
Other than that, Takara offers titer kits like BacPAK™ Baculovirus Rapid Titer Kit or BacPAK™ qPCR Titration Kit. The rapid titer kit works similar to the plaque assay, however the cells are fixed and stained for gp64 after infection. I tried this one out and it works well, however i recommend you use the suggested improved fixative to get some nice results.
The qPCR kit simply quantifies the amount of baculovirus DNA you have against a standard with known titer. While very straight forward keep in mind that the amount of DNA does not necessary correlate with the real number of infectious particles in your solution as you do not have a biological readout here.
Best of luck to you!
Hello Todor,
I appreciated for your answer. I have a small break for the experiments, so I answered late, sorry.
Those are quite useful information, thank you. I will try it.
Best regards,
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