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How to choose the best LPS concentration to stimulate macrophages? - (Sep/11/2007 )

Hi, I wanted to study the production of some cytokines in LPS-stimulated RAW264.7. To determine the best LPS concentration to work for, I performed LPS titration by stimulating the cells with increasing concentration of LPS followed by ELISA on the supernatant collected.

My questions are, which of the concentration that I should choose? Is the the lowest LPS concentration that stimulated the maximal response (highest level of cytokine produced) or the concentration that stimulated half the maximal response, etc etc?

For your information, I'm going to test my compound of interest whether it can inhibit the cytokine production in the LPS-stimulated macrophages.
Thanks

-money-

QUOTE (money @ Sep 11 2007, 07:37 AM)
Hi, I wanted to study the production of some cytokines in LPS-stimulated RAW264.7. To determine the best LPS concentration to work for, I performed LPS titration by stimulating the cells with increasing concentration of LPS followed by ELISA on the supernatant collected.

My questions are, which of the concentration that I should choose? Is the the lowest LPS concentration that stimulated the maximal response (highest level of cytokine produced) or the concentration that stimulated half the maximal response, etc etc?

For your information, I'm going to test my compound of interest whether it can inhibit the cytokine production in the LPS-stimulated macrophages.
Thanks


Dear Money,

Your one step ahead of yourself. The first thing to do is to optimise WHICH LPS TO USE i.e. from E.coli/S.typhosa etc etc. Each individual LPS will stimulate the cells to a lesser or greater degree. In our lab, Salmonella Typhosa gives the best response.
Once you have picked which LPS to use, THEN do a dose response curve. The range I use is for the stimulation of Nitric Oxide synthase and not for ctokine production. This is from 1ng/ml upto 10ug/ml.
Once you have done this, if I was doing your experiment then I would use the dose that gives 50% stimulation. Then do a dose response curve on each compound. If it is drug discovery you are after then this will be just an initial screen.

Regards

Rhombus

-Rhombus-

Hi Rhombus,

Thanks for your reply. Yes, what I am doing is just some preliminary testing on my compounds.

Sorry but may I know what do you mean by "best response"? Is it the type of LPS that produces the higher response compared to other LPS? In fact I'm a bit embarassed to ask but besides to get the best response, are there any other rationales behind using different types of LPS for stimulation? I have actually tried using LPS from E.coli and Salmonella minnesota.
Thanks.

-money-

QUOTE (money @ Sep 12 2007, 05:58 AM)
Hi Rhombus,

Thanks for your reply. Yes, what I am doing is just some preliminary testing on my compounds.

Sorry but may I know what do you mean by "best response"? Is it the type of LPS that produces the higher response compared to other LPS? In fact I'm a bit embarassed to ask but besides to get the best response, are there any other rationales behind using different types of LPS for stimulation? I have actually tried using LPS from E.coli and Salmonella minnesota.
Thanks.



Dear Money,

I am measuring the cells response to increasing doses of LPS. The marker I use is the induction of Nitric Oxide synthase. The " best response for me " is the LPS which gives maximum expression of this enzyme AT THE LOWEST CONCENTRATION relative to the source of LPS.
In my hands S. typhosa gives a larger expression of iNOS at 1ug/ml than E.coli at 1ug/ml.

For other researchers the type of LPS used maybe important to them for other reasons i.e. the mechanism of action might well be through different secondary messenger sytems.

Hope this is useful

Regards

Rhombus

-Rhombus-

Thanks Rhombus

-money-