I am trying to detect cytokines TNF and IL1B in primary rat glia culture media (high cofluency, no phenol red) after 40 min treatment with amyloid b oligomers. Unfortunately I could never reach detection limit (15 pg/ml). I could also not detect anything in lysed cultures (hypotonic buffer) and after 10x concentration of cell culture media. I would be very grateful if somebody could share their experience in ELISA-based detection of cytokines in cell culture media.
Hi Katarzyna,
I usually measure cytokine responses in cell cultures like PBMCs, T cells, macrophages, but not in glial cells. However, from your question I wonder if your stimulation experiments with oligomers have any positive controls (for example, LPS stimulation)? If so, did they also not show any detectable cytokine levels?
That would help you find out if your cultures are able to respond to any stimuli. From there on, you can continue troubleshooting your experimental and detection systems. Hope others with specific experience on primary glia cell stimulation experiments can provide more input.
Best,
Ricardo
Do your primary glial cultures contain both microglia and astrocytes? Or just one type? This may determine the response you see.
I would definitely agree with Ricardo that you should include a positive control like LPS (at least microglia will definitely respond) so that you know if it's the case that your cells really just aren't responding to the amyloid b oligomers, or if it's a more intrinsic problem.
The other problem could be the time course--IL1b protein should be up by 1-2 hours, but maybe not too much before. I'm not as familiar with the TNF protein time-course, but I think it should be before IL1b. Anyway, it might help to try a 2 hour time point.
Best,
Jessica
you can also try concentrating the media with Millipore amicon concentrators, once you have a positive control and know your assay is working appropriately.
Dear all, thank you for suggestions. I have no microglia in my culture only astrocytes. could anyone indicate me how long and what concentration of LPS should work? I would be very grateful.
I'm not as familiar with astrocyte immune responses, but I found this paper (http://www.jimmunol.org/content/150/7/2659.short) that suggests that LPS might not be the best positive control for astrocytes, since the authors found no response to LPS, but a large response to IL-1b. So you may need to add cytokines to the culture, instead of LPS, and it looks like they had to wait 16 hr to see peak protein levels of TNFa at least. Good luck!
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