I am treated PBMCs with various compound to observe cytokine expression using ELISA, however, I am getting high expression in my untreated control cells as compared to my media-only control. I am using frozen PBMCs which are thawed, washed and rested over night in RPMI at 37C and 5% humidity. The cells are treated for 24 hours in the same conditions then cell culture supernatants are collected and run in the ELISA. Are the certain conditions are stresses that may induce cytokine expression in untreated cells?
Could you clarify what you mean by "untreated controls" vs. "media-only control"?
Have you checked for microbial contamination? This could cause higher cytokine expression. Maybe you only have it in a few wells?
Do you keep your treated cells in close proximity to your "untreated controls"? If so, perhaps some of the stimulus may have fallen into neighbouring controls causing a response.
Have you repeated this experiment more than once? If not, I would recommend doing so to see if this is a one-off event or if this keeps happening consistently.
Victor, I have repeated this multiple times and still get high cytokine stimulation in my "untreated controls" (PBMCs in RPMI). Contamination is a good idea to check for.
Francis, I am unable to use fresh cells since I am working far from any supplier or research hospital, so I am trying my best with the frozen cells. Have you seen any increased expression with frozen vs fresh?
I think if you're problem was due to the cells being frozen, you would expect to find it in all the samples, not just the untreated controls.
I think you should still specify what the difference is between untreated control and media control. You said your "untreated control" was PBMCs in RPMI...so then what's your media-only control?
Thank you for your suggestions! My treatments are also very high along with the negative control, so I worry it is something about cells or overall cell culture process I am using. The media only control is just RPMI, that's low so I know its not an issue with the ELISA procedure.
Freezing and thawing affect protein likewise expression. It is possible that the storage affected the cytokine yield.
As Francis said, you colud try with fresh PBMCs. Maybe your own PBMCs. All you need is small blood sample and ficoll paque or something like that.
Does your standard curve work well?
I agree that contamination could be an option.
Are you 100% sure that you are not stimulating with aCD3/CD28 or IL-2 for example? This is very obvious, but you don't lose anything checking.
Eduardo, thank you for your ideas. How would I check for CD3/CD28 or IL-2 stimulation? The negative control cells that are highly expressing are just cells in RPMI.
Ok, that is what I meant. I thought that maybe you were incubating your control with antiCD3/CD28 or IL-2.
One question, what about the cytokine levels in your treated samples? Is it higher than your untreated control? or are they the equal?
Roughly equal, that's the problem, although those treated with LPS are roughly 25% more. I should be expecting higher than that, so I'm assuming that my baseline expression of the cells is just too high.
This comes Just from my ignorance but I don't remember that LPS was a good Cd4 T cell activator.
LPS activates DCs and macrophages. Maybe it doesn't work well with Cd4 t cells
Changes in the Cytokine expression of the untreated controls is to be expected as they are live cells being influenced by their environment. The treated samples are subjected to the same pressures plus your stimulation protocol. The treated and the untreated should be matched so the untreated represent your baseline, under these conditions it should not matter how much the profile changes as you will be only noting the treated changes relative to the controls
Natasha, this article might be useful to you Article Isolation And Preservation Of Peripheral Blood Mononuclear c...
Basal activation of your cells might be related to release of different molecules due to cell death. DNA, RNA, but also ATP and other nucleotide, which can signal through purinergic receptors in monocytes (macrophages) are well characterized molecules that activate this cell population resulting in cytokines secretion.
Although not related to your basal cell activation it is interesting that ATP also is secreted during CD4+Tcell activation giving an important autocine signaling to T cells.
In this context, longer resting times with careful wash can be useful. Also, activated monocyte separation, by adherence, can be useful.
Depending on the length of your experiment location on the plate can influence activation of PBMC. Wells along the edge of a 96 well plate, and the corner wells in particular (where people often put controls) can evaporate more quickly than other wells on the plate, this can lead to stress which can activate the PBMC. If your stimulation is more than a couple of days it is worth filling the outer wells with PBS or DI water. You can also just vary the location of your negative control well to see if that changes anything.
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