I am evaluating the anti-inflammatory effect of plant extracts and performed the experiment many times using RAW 264.7 cells but could not get the significant results. I want to know that how much cells I must take for NO experiment i.e. 2x10^5 or 3x10^5 or any other number of cells??? What are the possible problems which should I overcome to get the significant results?????
Every single effort will be highly appreciated.
Sincere Regards,
Hi, Zaheer -
Can you give us a few more details? For example, how are you measuring nitric oxide production? When you say you don't 'get significant results', what does this mean? For example, do you detect nitric oxide in your controls, but don't see less with the aniti-inflammatory agents; or do you not detect nitric oxide at all?
(I haven't worked with these systems for a long time, so I may not be remembering well, but you may need closer to 10^6 cells)
If cell count and culture protocol are of your concern, please see the following link as the culture procedure for RAW 264.7 provided by LIPID MAPS.
http://www.lipidmaps.org/protocols/PP0000000101.pdf
they mentioned that using 24-well plate seeded with 2 x 10^5/well, wells will reach confluence in 30 hours.
The first question I would ask me is:
1. My system is releasing nitric oxide ?
2. If the answer is yes; then.... how sensitive your method to detect its metabolites: nitrates / nitrites
3. Quantification of nitric oxide may do, so only with a specific electrode; if you do not own...
4. Measure the production of nitrate / nitrite: either.... spectrophotometric assay or by a commercial kit.
5. if you work with, an in vitro experiment ... no problem... there are many commercial kits for this purpose .....
6. If you want to do for a biochemical assay .....this is easy ( and less expensive) , and can be found online methods....
We have used both J774 and RAW cells in our experiments and the most important thing to assay first is the source of your LPS. In our hands LPS from W.S. Typhosa was the most potent inducer of iNOS in both cell systems. You can get induction from using 10ng/ml upto the maximum of 10ug/ml. However at the upper limit so much NO is produced that you find that after just 24hrs most of the cells are dead or dying.
The number of cells in your 6 well plates should be at the 10^6 level to give you the ability to measure the Nitrite in the cell culture media. Remember to use DMEM as this has no background Nitrite....FCS does have small levels of Nitrite but this can be dialysed away.
The other trick is to induce in arginine free conditions...therefore the LPS will induce iNOS but with no arginine NO will not be producd to kill the cells. After a 24 hr induction the induction media can be washed away and replaced with normal DMEM with arginine.
I hope this helps a little
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