I am planning to run a Luminex assay using cell supernatant to assess cytokine secretion by tumor cells.
I came across different methods in literature; some suggest seeding in complete media and then replacing it with serum free media for 24-48h. While others suggest seeding in low serum media (5%) for 24-48h.
Confused which one to follow!
Please advise.
Hey Tanvi,
it highly depends on your experimental setup. What cells do you use? What kind of stimulus do you use. Do you know that the stimulus will induce production of the cytokines, chemokines or growthfactors you want to measure in your Luminex.
As an example we measured cytokines, chemokines (and so on) in supernatants from cultured macrophages infected with bacteria as well as plasma samples from infected mice in one Luminex assay (meaning one plate). The Luminex assay is just the method for detection. So it does not really depend on serum or no serum (only if you want to study starvation conditions in cells), but more, if you know that your cells will respond to a certain stimulus. If your cells respond to starvation, then it makes sense to remove the serum. For the assay itself it is of no importance at all. Perhaps you can give me a more detailed experimental setup for further advice.
All the best and stay healthy,
Marc
Hi Tanvi,
The serum may contain cytokines which might be detected or which may act on the cells.. but you need to also consider that starving the cells of serum may have a negative impact on the cytokine release. In this regard, I recommend that you use the 5% serum culturing conditions and that you make sure to use a control consisting of the 5% culture media alone, without cells. Its not perfect, but you can at least assess for cytokines present in the 5% serum this way.
Hello Mark,
Thankyou for your answer.
I am not using any kind of stimulus. The question of interest if what cytokines/chemokines are secreted by tumor cells (14 syngeneic mouse cell lines).
Also, the seeding concentration of cells would vary since all of them grow at different rates. However, I am making sure that the confluence is consistent and plan to harvest the medium at 70& confluence for each cell line. I plan to culture the cells in 1% FBS without antibiotics.
Hope this will work. Please feel free to add an input.
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