I used the LPS to induce the U937 monocyte cell line. But I didn't know anything about the activity of LPS (good or degrate) because the U937 did not translocate of the HMGB-1. I would like to the use the cheap method for checking activity of LPS. Thank you.
W. Dankai
Dear Wiyada, I have experience with LPS and human monocyte (natural human monocytes but not U937 cell line). For LPS activity I checked LPS inducing TNF-a secretion by monocytes. I know U937 produce and secretes cytokines such as TNF-a , IL-6,...
It means you can add LPS with different concentration to the cell line culture media and then measure e.g. TNF-a secretion in culture media by ELISA method. And also comparision with blank or control cells.
So a simple method is cytokine secretion following LPS induction.
best
mehdi
There is this Limulus amebocyte lysate assay to measure LPS activity in a given solution. It is from sigma as far as I remember!
E8029 Sigma
E-Toxate™ Endotoxin standard
for endotoxin quantitation
This is not the assay, they mention it in this page but I cannot find it anymore. Perhaps some other brand may sell it!!
http://www.piercenet.com/product/pierce-lal-chromogenic-endotoxin-quantitation-kit
http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_QCL-1000_Product_Insert.pdf
You can verify LPS activity by assessing nuclear translocation of NFkB by immunofluorescence at various timepoints after treatment. NFkB translocation is oscillatory so there will be significant heterogeneity among cells with positive signal in the nucleus. 60 minutes should be the first peak and so most of the cells should be synchronized at that point, but at 120 it will probably be about 10% and at 180 minutes it will probably be about 50% with positive signal. I've found 60 minutes to be a good indicator.
Thank you all. Mehdi Hedayati, Pavel Montes de Oca B and Jordan Robin Yaron .
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