Hello,
I am trying to show production of IL-1B in BV2 cells activated by LPS+Nigericin.
Tried seeding all the ranges from 2*10^4 - 10*10^4 cells per well in a 96WP.
Treated with LPS (1ug, 2ug, 3ug/mL) for 4hrs.
Treated with Nigericin (5mM) for overnight.
Collected supernatant and tested for IL-1B using ELISA.
I am not able to see any IL-1B levels. Could anyone tell me what is it I am doing wrong here?
Thank you.
Ramya
You are using high doses of LPS which should induce cytokine production-- perhaps could even be toxic. Have you performed a viability test on your cells to ensure they are surviving the LPS challenge? Four hours is also a short time period-- you may consider looking at 24hrs post LPS challenge. Using 10ng/mL LPS, I observe minimal IL-1B expression in media of Primary and Immortalized microglia after 24 hrs (See Fig 1 in attached paper). You may want to consider looking at a different cytokine (TNF-a or IL-6 ).
Also, the strain of E Coli your LPS comes from could be affecting the response. This strain from Sigma works really well for primary and immortalized microglial/macrophage cell lines: E. coli O55:B5; Sigma, cat# L2880.
Good luck with your experiments!
I agree with Elliot J. Glotfelty. Try first to obtain a positive response to LPS dilutions after you assured the viability of your cells.
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