I have problem to detect different cytokines in supernatant after the treatment of cells with LPS or TNF alpha by ELISA.
Can you suggest me the way for concentration cytokines in cell supernatant or some more sensitive method for cytokine detection?
Thank you in advance.
There is really no method to concentrate proteins, unless you want to do a size separation column. But i don't think anyone goes to these lengths for supernatants.
There are ultrasensitive immunoassays that have lower limits of detection. You will have to see which ones that will work for you.
We use luminex for cytokine detection.
A bead based format can also be used with flow cytmetry to do cytokine quantification.
ELISAs these days are highly sensitive (LOQ 8 pg/mL). What is the limit of detection for your ELISAs? What cells are you using?
LPS induces a ton of cytokines, even more than TNF. So if you are having issues with detecting cytokines when using LPS, I don't think a different method of measurement would help. Flow cytometry won't work as it requires cells. Bead based immunoassays like CBA and Luminex have comparable sensitivity to ELISA and the cytokine values are usually identical when using the two methods (ELISA is even better in come cases). The only exception would be if you are using in house ELISA reagents. If that's the case, try commercial ELISAs and see if that helps. I have had good experience with BD OptEIA kits.
If you are using a commercial ELISA already, I would look at the experimental setup more closely. Like Shen-An Hwang said, there is no need to concentrate sups when using strong stimulants. If you are not able to detect at least 20-100 pg of any cytokine, then either your assay setup needs tweaking or your cells are not responsive to the stimulant.
Dear there is no need to concentrate the supernant cytokine because the ELISA will be sensitive to detect it.
Julie Joseph thank you for the answer.
I used commercial ELISA kit from PeproTech and detection limit should be between 16 and 8 pg/mL depending of type of measure cytokine . The cells I was treating were primary trophoblast cell line ACH-3P and third trimester trophoblast.
Maja Gajic, I haven't worked with trophoblast cell lines and so can't provide much guidance. Usually, IL-6 is a good indicator of whether or not your assay setup works when using LPS. Try looking for cytokines that you know should be expressed at high levels by stimulated trophoblast cells and see if you can pick those up. If not, you may need to optimize your assay setup (esp. the cell numbers, the stimulant concentration and the stimulation time frame) to get optimal results. If you can, then that means your cells are secreting some cytokines but not others.
As I said earlier, I think your problem lies with your assay setup not with ELISA. If you would like to rule ELISA out as the problem, stimulate some macrophages (primary/cell lines like J774/Thp1) or spleen cells with the doses of LPS/ TNF you are using, harvest sups at 6-24 hours and do ELISAs. If you see cytokines there, that is definite proof that you need to further optimize your trophoblast culture/stimulation conditions.
In addition to the suggestions that ELISA will be able to detect cytokines without prior concentration, you can still concentrate your samples by: (1) ultrafiltration; (2) freeze drying; (3) dialysis vs dry PEG. Just note the MW cut-off for the filtration and dialysis membranes and your cytokines.
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