cell culture for ELISA

[mywillowtree]

Hi all!

I'm trying running a ELISA with cell culture supernatants, and my results are all over the place.
I'm pretty confident in my Elisa - its a commercial precoated kit and my standard curve was fine - so that only leaves errors in the culture technique.
What could I be doing wrong that would lead to inconsistant results in the ELISA?

I'm using 16HBE cells (adherent), and testing PGE2 levels after stimulation and inhibition. Supernatants are collected after 4 and 24 hours
If you would like more specific details, let me know!

[danfive]

Originally Posted by mywillowtree

Hi all!

I'm trying running a ELISA with cell culture supernatants, and my results are all over the place.
I'm pretty confident in my Elisa - its a commercial precoated kit and my standard curve was fine - so that only leaves errors in the culture technique.
What could I be doing wrong that would lead to inconsistant results in the ELISA?

I'm using 16HBE cells (adherent), and testing PGE2 levels after stimulation and inhibition. Supernatants are collected after 4 and 24 hours
If you would like more specific details, let me know!

Are you talking about conditioned media or supernatant of cell culture lysate?
Either way 1st thing will be to do a dilution series. Analyze the results: Random signal up/down = interference, hook effect = protein associating together at high density, no signal = inhibition (could be due to pH)

[Jenniferayt]

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[Jenniferayt]

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