Previously, we could not detect any cytokines. We suspect that supernatant is dilute due to few cells in culture. (Increasing cell density is not an option for us.
Hi, just freeze-dry your samples ( for 10x or more concentration), avoid serum albumin or remove it with columns and make sure to take the overall cell number and death-rate into account of your cytokine-concentration calculations..
You could try doing the capture step for longer (24h) at 4oC to increase sensitivity, or if you have the vacuum plates with your luminex kit you could add the unknown medium a number times (or as a larger volume) to capture the maximum amount of cytokine in your fluid.
With amicon ultra filters you can concentrate your supernatant, very easy and fast method.
Filters are the easiest, but do a few preliminary experiments to make sure you understand what sort of loss you may be experiencing. Even though you are concentrating most of the cytokine, you may be losing some on the filter at the same time.
For ELISA, you could try changing the colorimetric substrates to more sensitive chemiluminescent substrates. or you could ask a CRO to detect using AlphaLISA/ TR-FRET assays, which are more sensitive then traditional ELISAs.
Please check this:
http://www.novateinbio.com/en/content/70-alphalisa-and-alphascreen-assays
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