Hello everyone. I want to do an experiment to assess the immunogenicity of a Salmonella typhimurium vectored DNA vaccine in bovine PBMC (Peripheral Blood Mononuclear Cell). I’m not sure on various steps involved. I would like to ask if anyone has a protocol that gives details on this.
Thanks.
Dear Azar,
so, you want to use a GMO live Salmonella to transport foreign DNA into bovine PBMC (I guess mainly monocytes), correct? If so, any MOI between 0.1 and 100 has been used, but the higher your MOI, the sooner have the bugs killed the cells. If you use an MOI of 1, that should do the job, but you need to kill the bugs after 2h by addition of gentamycin. When do you want to look for expression of your gene? Bovine PBMC survive up to 3-5 days quite well, depending on their activation stage. If you want to know whether the bugs are inside or attached to the surface, that becomes a bigger challenge.
Hope this helps a bit
Kind regards
Dirk
Dear Dirk,
Your help was very useful for me and I really appreciate you for your kindness.
I use a live attenuated salmonella typhimurium as carrier vaccine. I want to measure IFN-γ production in cell culture supernatant 18 or 24 h after culture stimulation.
I don’t know about suitable time for stimulating of PBMC after cell transfer to 96-well flat-bottom plates and the concentration of gentamycin that I need to add cell culture. Would you please guide me about these things?
Dear Azar,
if you use a live attenuated Salmonella, you will measure IFNg after these timepoints, but your bug might still outgrow/overgrow the cells. I would do earlier timepoints as well, such as 2h, 6h, 12h. But: do you use cells from naive or immunised/infected animals? I would be not surprised if your control (the live attenuated Salmonella strain without the vaccine DNA in) would induce similar amounts of IFNg. Regarding the gentamycin etc - please check the literature - if you search PubMed for Salmonella bovine vaccine, you get loads. Start by looking for Villarreal-Ramos B. They did loads of work on Salmonella exposure of macrophages/DC in the bovine system.
Can I ask a different question - have you ever worked with bugs/bovine ells before in vitro?
Dear Drik,
Thanks a lot for your reply. I really need some help for my work because this time is the first for me to do cell culture and check the immunogenicity of vaccine!
I construct a DNA vaccine against paratuberclosis and I want to use Salmonella as a career for checking the immunogenicity on PBMC of infected and naive cows. I think it’s better to check it in vitro, but now I have some problems!
You said that you would not be surprised if the control, induce similar amounts of IFNg. Why you think like this?!
If you have any suggestion for checking the immunogenicity of this vaccine, tell me please.
Dear Azar,
there are differences between life and dead Salmonella what cytokines/amount of cytokines released by cells, but nonetheless, your Salmonella (even as attenuated one) will induce a IFN response in bovine PBMC. This is just how the cells respond, normal immune reaction. And it may not be that your DNA vaccine is inducing a very strong additional response.
Have a look at this PLOS One paper:
http://www.ncbi.nlm.nih.gov/pubmed/23950909
Hope that helps?
- Badges
- Science topic