Dear Finny,

While I have not stained C6/36 cells before, I have an idea what your problem might be. Arthropod cells are often persistently infected by viruses - For example Aag2 cells are (if I remember correctly) infected with Cell fusing agent virus (a flavivirus) and many tick cell lines are infected by orbiviruses (Bell-Sakyi and Attoui et al 2013). Both of these types of viruses would give you a positive staining for dsRNA.

I believe C6/36 cells are infected by densoviruses, which are not RNA viruses and would not explain your non-specific staining, but I think they might be infected with other 'endogenous viruses', which could have dsRNA as part of their genome or replication. I think they just haven't been investigated in enough detail.

Maybe you could test a completely different antibody (against a cellular protein) to test your protocol and if it works for a different antibody, your 'non-specific' staining could be explained by the detection of viruses present in these cells before you infect them with CHIKV.

Just a thought, best of luck,

Claudia

Thank you Claudia. I had already thought about this as a possible explanation. It might mean that I might have to use a different batch of the C6/36 cells. Staining with anti-CHIKV-envelope protein 2 did not give any non-specific staining. But I am working on solving these problems.

It might even be that C6/36 are all infected and a different batch may not help you (they might have been derived from persistently infected eggs/larvae back when they were made), but good luck for solving these problems, I hope you can get your data somehow! :)

Infact, I even asked members of Dr. Kohl's lab in Glasgow for their protocol. It somehow seemed to work for them. So, if this problem continues, I might just ask them for the cells.

Ah that's good. I assume Alain will probably be happy to send you the cells - but if it's not C6/36 cells in general that are the problem, then maybe it could be your blocking reagent. Have you tried different ones? We normally use Cas-Block from Invitrogen (and Alain's lab probably does as well) - it could be that something in your FBS is acting as a non-specific binding site for the J2 antibody. I believe you can get these artifacts from different BSA batches/milk powder etc ... anyway, I'm probably not telling you anything new, so good luck!!

hi Finny, Long time.

My lab works on Chikungunya virus and I believe we have the right recipe for it. We do not have extra BSA blocking step. But the difference is that we use in-house raised antibody against E2 region of CHIKV.