We understand that different types of data can be gained from the various options we have for collecting cell types and cytokine profiles from our pre-clinical groups (vaccine testing), and that ideally we would test multiple ways and compare. However, as funds are not endless and we must make a decision, I am hoping some of you could provide insight on what our best way forward is.
My understanding is that peripheral cytokine levels may be low, and could be increased by boosting 24-48 prior to collecting and using plasma, however, with our protocol boosting is not optimal (the organisms are infected). We can, however, take a blood sample in heparin. Would we likely pick up the cytokines this way? Should we consider ELISA over Flow cytometry or vice versa?
We are planning to run a cellular assay using splenocytes, and stimulating them with the main antigen included in the vaccine. Would we be better off testing the cytokines from these assays?
Would the cellular data through flow cytometry here be as useful, as it seems less direct and subject to false replication of the in vivo response.
Thank you!
Blood: ELISA would offer the most reliable way of analysis, and probably the most direct ex vivo as you can get.
For the splenocytes: No reason you couldn't do a combination of intracellular cytokine (ICS) and ELISA. Just pellet you stimmed cells and collect the cell fraction and supernatant after restim. ELISA will allow you to easily quantify released levels of cytokine, ICS will pin point which cell types are producing it. RT-qPCR provides a similar answer to ICS, but some reviewers believe that transcripts =/= protein.
RT-qPCR (reverse transcription-quantitative PCR) may be an option for you... if you have the expertise in place. One-step (RT-qPCR) analysis of total splenocyte RNA extracts could provide you an alternate avenue of exploration. If you already know that increased expression of specific cytokine mRNA is correlated in kind with increased [cytokine] protein production, this is a key way to make your case. If the infected "organisms" you allude to are rat, mouse, human, dog, or zebra fish, there may already be some pre-designed assays available. Funding is always a problem isn't it?
Thank you Jack, that is a good idea. I don't have a lot of experience with RT-qPCR but a colleague does...thus this may be a viable alternative!
What is your main question: level of cytokine production or secretion? If you would like to estimate the level of cytokine production and you think that it will be low, than your method is qPCR (as was mentioned already). If you would like to know the "real"level of cytokine secretion, I would use an ELISA kit (R&D systems has some) and fresh material (freeze and thaw affects activity a lot). Success!
Blood: ELISA would offer the most reliable way of analysis, and probably the most direct ex vivo as you can get.
For the splenocytes: No reason you couldn't do a combination of intracellular cytokine (ICS) and ELISA. Just pellet you stimmed cells and collect the cell fraction and supernatant after restim. ELISA will allow you to easily quantify released levels of cytokine, ICS will pin point which cell types are producing it. RT-qPCR provides a similar answer to ICS, but some reviewers believe that transcripts =/= protein.
For selected cytokines, there exist MACS Cytokine Secretion Assays (Miltenyibiotec.com) designed for highly sensitive detection of viable cytokine-secreting cells. The cell membranes are covered by anti-cytokine antibodies which capture secreted cytokines. They are then visualized using a secondary antibody by flow cytometry. You can even magnetically separate these cells from suspension for further analysis.
I am in the vaccine research field and in our group we choose determining antigen-specific immune responses correlating with the cell type responsible by employing intracellular cytokinne analyses with flow cytometry; yes, this is expensive but tells which cell type is responding and could assist in improving the vaccine formulations using appropriate addditional agents (e.g. adjuvants with ability to scew towards cellular vs humoral; Th type etc). Alternately, we use cytokine-ELISPOT analyses to detect total IFNg secreting cells but also collecting supernatants from this assay to detect other cytokines by cytometri bead array (to detect multiple cytokines up to 20+) in as little as 10-20ul; in addtion, from the ELISPOT assay, cells can also be collected for RT-PCR: thus, multiple readouts can be obtained with one set of cells (we choose this because many times the cell yeild with biopsy samples in humans and or nonhumman primates we work with is limiting to allow other detailed assays). Hope this long winded answer is useful
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