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If I want to study the activation of dendritic cells (human blood monocyte derived) by antigens, which markers should I focus on? The ultimate objective is to study T-cell proliferation post-DC activation and antigen presentation. But at first, I want to show DC activation occurs before overlaying T-cells. What assays can I perform to show 'DC activation' besides target marker transcript (qPCR) expression, protein (ELISA) expression, flow cytometry for the markers?
Links to original articles for citation will be much appreciated.
Hello Farah Shahjin
You can refer to the research article below for more information.
Article Activation of human monocyte-derived dendritic cells in vitr...
Activation of DCs was determined by assessing the expression of co-stimulatory and maturation markers (CD40, CD80, CD83, CD86 and HLA-DR) by flow cytometry, and production of cytokines (IL-1β, IL-6, IL-10, TNF-α, IL-12p40, IL-12p70 and IL-2) by ELISA.
DC function was determined by assessing their ability to activate naïve T cells. Activation of T cells was assessed by measuring cell proliferation and cytokine production.
I hope this is helpful.
Best Wishes.
Hello,
As suggested, I would do an easy assay with this panel: live/dead, cd80, cd86, mhc II and check the upregulation of these markers by flow. You have to make sure your cells are not over activated by your compound (they will die if you over activate them). You could also add CD11c to show that your differentiation went well. The ELISA on IL-6 will also give you a strong indication of activation. Remember to use LPS as positive control in these assays.
You wrote ''what can i do beside RT-PCR, Flow and ELISA''...I guess you should do these 3 assays for sure since reviewers will ask for those. In my opinion, flow is way better than RT-PCR since mRNA expression does not alwais mean actual protein expression and the timing in the upregulation of the mRNA transcript happens faster compared to the secretion of factors such as Interleukins.
Regards
Francesco
Thank you Francesco and Malcolm for you prompt replies!!! Much appreciated!!
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