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Measuring nitrite from LPS-activated RAW264.7 cells - (Aug/14/2005 )

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QUOTE (Rhombus @ Feb 26 2007, 11:00 AM)
QUOTE (IMMUNOSP @ Feb 25 2007, 09:44 AM)
QUOTE (Rhombus @ Jun 8 2006, 11:53 AM)
Dear Lab newbie,

You should have no trouble in measuring NO2- form activated RAW Cells, after 24hrs levels should be in the 10-30uM range.
It is true that different LPS sources induce iNOS with varying degrees, we have used salmonella Tyhphi LPS in our lab over the past 15 years. However there is evidence that contaminating levels of endotoxin can inhibit the induction of iNOS. So check that the endotoxin levels in your FCS are lower than 0.1ng/ml.
Also are your cells Mycoplasma tested, if they are not TEST them.
Also make fresh stocks of your LPS i.e weigh out the appropriate amount and make your dilutions. DONOT solubilise all the LPS and aliqout.

I used to make iNOS for drug discovery at Wellcome and Glaxo.
Best cells to use are J774, use LPS and IFN (10ug/ml/50units/ml) for maximum activation.

Hope this helps

Hi Rhombus,

Is there a big difference between fresh or freeze LPS?. I did a LPS stock, aliquoted it and froze it at -20ºC like some autors and SIGMA-ALDRICH web-page suggested (
Do you think it doesn´t works weel?.


I have been stimulating J774/RAW 264.7 for 20 years. I ALWAYS prepare fresh LPS from the lyophilised LPS powder. DO NOT dissolve the entire vial and aliquot. ALWAYS keep your powder at -20oC and when you weigh some out, do it quickly and immediateley return it to the freezer

Thanks Rhombus. I´m going to do in that way.


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