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

Hi, all:

Our lab is trying to measure nitrite produced by LPS-activated RAW264.7 cells as this seems to be a fairly common measurement seen on numerous research articles.

However, we came across the problem in that LPS didn't seem to activate the cells at all. Fllowings are what we did in the experiment and I would highly appreciate any experts experienced with this assay could help shed some hints to us since we've been stuck on this assay for months.

[experimental setups]
1. Trypsine RAW264.7 cells, count and seed them (in DMEM w/ 10% FBS) at 2x10^5 per well (in a 24-well plate)
2. Add LPS (diluted in the above same DMEM) at final of 1, 0.5, 0.25, 0.125, and 0 ug/ml
3. 37C, o/n incubation in CO2 incubator
4. Harvest supernatant for Griess assay (Griess reagent system by Promega)
5. Read the plate at 550nm

We are sure the assay itself works fine, as we always included NO standards along with our samples and the standards always generate a nice dose-dependent curve.

Someone had told us that sometimes it matters as which LPS source was used to the experiment. The LPS we used was purchased from Sigma (L7261, from Salmonella typhimurine; lot#043K4094). I'm kind of embarrassed to ask this question since it seemed this assay should be rather straight forward to perform and has been used extensively in numerous research articles. However, we've been repeating this simply assay for months and are getting no where...

Any of your suggestion will be highly appreciated. Many many thanks in advance!!

[experimental setups]
1. Trypsine RAW264.7 cells, count and seed them (in DMEM w/ 10% FBS) at 2x10^5 per well (in a 96-well plate)
2. Add LPS (diluted in the above same DMEM) at final of 10-100 ng/well
3. 37C, o/n incubation in CO2 incubator (24-48 hr) time dependent NO amount
4. Harvest supernatant for Griess assay (Griess reagent system by Promega)
5. Read the plate at 550nm

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, I have cultured RAW264.7 cell for two mouths yet. but no good experiment results from my cell. would you like to give me some suggestion due to RAW264.7 can not produce increased NO in cell medium after stimulated by LPS plus IFN-gamma in different concentration for 6 hours , but the Griess assay show me no different between control group and stimulated group.
My experimental setups
1. RAW264.7 cells, count and seed them (in 1640 w/ 10% FBS) at 2x10^5 per ml (in 30mm plate)
2. Add LPS+ IFN-gamma (diluted in the above same 1640) at final of (100ng/ml+1U/ml;1mg/ml+10U/ml;10mg/ml+100U/ml)
3. 37C, o/n incubation in CO2 incubator (4-6 hr) time dependent NO amount
4. Harvest supernatant for Griess assay
5. Read the plate at 540nm
the endotoxin levels in my FBS are lower than 0.1ng/ml since I have used high-FBS. And no bubbles introduced during the pipetting/diluting process

It troubled me that is poor cell morphology and viability. They are adherent as they are supposed to be, but a mixture of elongated spindle shaped cells and circular cells that are usually granular looking. Some are large dome-shaped and dendritic looking even. They reach confluence very quickly although im very conscientious about splitting them at 80% confluence. They still look like that after several passages .Its exasperating. I understand granularity in most cells is not a good sign. Does this apply to a macrophage line as well? It is the reason of poor Griess assay data?

Thanks for paying attention to my long delineation.

Dear dandanzhang_9,

I sent you a private message yesterday, but forgot just one important point.
ALWAYS seed your plates one day and leave them overnight to adhere and equilibrate. THEN add your LPS/IFN Gamma cocktail.

Hi,
I thinks you slite modify their protocoles for NO estimation.
1. detached cells by tapping or pipetting media by 1ml pippete many times keeping in the mind less stress cells .
2. count them and plated them in culture plate for atleast 6h and then added LPS at different concentartion.
and finally collect supenatent at different time point estimate NO in the supernatent.
hope its will be work.
aal the best and good luck.
awadh

very thanks for warm helps from everyone paying close attention to my trouble and I get so many valuable notes in this experiment .
I have done experiment according to your suggestions,and fortunately ,NO in medium changed from 2uM to the top of 11uM after induced by LPS plus IFN-gamma in different concentration after 24Hr.The time of inducing RAW264.7 cell is an important thing.I will do western blot to ensure the result .
Thanks again!