Does anyone know which stimulus can i use to detect Il-10 and IL-6 on Caco-2?
I have cultured Caco-2 for 21 days in a 12-well plate and did the stimulation with (25 ng/mL IL1b, 10 ug/mL LPS, 50 ng/mL TNFa) for 48 hour and by ELISA (by Peprotech kit) I only see basal levels with no differences between stimulus and non-stimulus....
Hi Carolina,
I used Caco-2 just a couple of time in my previous lab, but I found increased levels of IL-6 upon stimulation with IL-1beta and TNF-alpha, as well as increased expression of mRNA. Did you replace culture media before stimulation using fresh medium? Otherwise excessive cytokine accumulation (after 21 days) into the medium avoids to evaluate difference upon stimulation.
You possibly should check: 1. cell "passage number": lower is better, of course. Cell ageing could interfere with cell signaling. 2. Twenty one days of culture sounds a little bit at the limit of feasibility (because of cell senescence process). 3. A suggestion: I used to stimulate epithelial cells in the absence of FBS or with low concentration of FBS, because in our experience, FBS intereferes with induction of cytokine expression.
Thank you so much Valentino Bezzerri for the help.
In fact, I guess it might be the passage number, once this cell line is getting old.
I don't think is what you refer as "Otherwise excessive cytokine accumulation (after 21 days) into the medium avoids to evaluate difference upon stimulation." because we don't get any IL-6/IL-10 concentration at all (for controls with and without stimulation).
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