Dear all,
I tear one's hair out with my experiment.
In the lab, we have validated whole blood activation with heat killed bacteria. we have seen an increase of inflammatory cytokine in monocytes (like IL-1B).
But for the project, we want to use PBMC and not whole blood.
2h after resting, I cultured my PBMC in PP tube during 24h with ou without heatkilled bacteria. 4h before the end I added Brefeldin A.
Finally, I have the same response with or without bacteria. I shown IL-1B, IL-8 and TNFa on CD14+ cells in both condition
Thank you for help.
Best
Ana
So... depending on the bacteria... it probably needed the complement and/or the antibodies present in the whole blood to enhance the cytokine production.
Without it... heat killing (56C or autoclaving?) sometime can take off a LOT of the cell wall components that makes interact with the PRR to initiate a good inflammatory response in monocytes/macrophages. 24 hours is too short for any T cell help... So... if you want to use PBMCs... my suggestion is to culture for longer than 24 hours...
And if you are seeing increase cytokine responses without any stimulation...then it's nonspecific activation due to the processing and/or contimination.
Agree. It is non specific activation , possibly due to contamination. CD4 stimulation usually require more than 1 day of incubation, may be 2-3 days. If I were you I'd try 36, 48, 60 hrs. fore the same sample and protocol.
It should definitely work. Reading your comment, I assume you are measuring it by flow cytometry, right? Maybe you are adding the Brefeldin A too late and you are missing the peak of cytokine production. If possible, you could try to stimulate your cells with the HK bacteria and collect the supernatant after overnight incubation. The proinflammatory cytokines should be in the supernatant. You can also use LPS as a positive control (especially if you are interested in Gram-negative bacteria).
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria