I'm struggling a bit with the TNFalpha assay using RAW264.7 cells:
1. LPS is leading to only an 80% increase in TNFalpha mRNA levels, which seems low.
2.Increasing the dose of LPS leads to a reduction in TNFalpha induction (very weird)!
I am thinking that there is a problem either with the LPS I am using(it is a bit cloudy in “solution”, currently I am using L6529 LPS from Sigma) or perhaps I should be using ELISA rather than qRT-PCR.
Any thoughts/suggestions would be helpful.
Thank You very much!!!
I just happened to know the answer as I did a one year sabbatical in Brisbane in Jenny Stow's lab, and she works extensively with RAW cells.
LPS from E. coli should not be used as it activates both TLR2 and TLR4 receptors. So your signalling will be a bit messy. Jenny's lab always uses Salmonella LPS which only activates TLR4.
We usually use 100 ng/ml LPS for all our experiments on TNF release from RAW cells. You can check our paper attached to this message.
We always used ELISA or flow cytometry to measure TNF levels in supernatants from these cells. I think it's a bit more straightforward to do ELISA rather than qRT-PCR but that's up to you.
Hope this helps.
I just happened to know the answer as I did a one year sabbatical in Brisbane in Jenny Stow's lab, and she works extensively with RAW cells.
LPS from E. coli should not be used as it activates both TLR2 and TLR4 receptors. So your signalling will be a bit messy. Jenny's lab always uses Salmonella LPS which only activates TLR4.
We usually use 100 ng/ml LPS for all our experiments on TNF release from RAW cells. You can check our paper attached to this message.
We always used ELISA or flow cytometry to measure TNF levels in supernatants from these cells. I think it's a bit more straightforward to do ELISA rather than qRT-PCR but that's up to you.
Hope this helps.
Paige suggestion is very useful, your description of results remind me what happens sometimes for mRNA after over-night or longer periods of stimulation.
Hey Klaudia,
also keep in mind the different time point of TNF measurement. After LPS, TNF protein peaks after 4h. So if you measure later, i.e. after 24h you might lose the signal.
Good luck and check out this paper
http://www.ncbi.nlm.nih.gov/pubmed/16506285
regards
Simone
Hi Klaudia,
I am unable to understand the level of TNF is 80% to what????
Reactive nitrogen species could also account to cell toxicity and change gene expression. Maybe it much better to catch expression on mRNA level before inducible NOS start working (4-6 hr).
Dear Klaudia,
The LPS untreated RAW cells will have very less level of TNF expression which must will increase upon LPS treatment.
In this setup, if you measure protein level by ELISA it would be better. I have used 1µg/ml LPS (same as you used) and detected TNF upto 4 h. You can follow th link.
http://www.ncbi.nlm.nih.gov/pubmed/23266381
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