Using LPS stimulation, I could easily detect iNOS induction from Macrophage (Ma) or Microglia (Mi) after 6 hr, 16hr by qPCR.
Even though I detected the translocation of NF-kB p65 into the nucleus after 30 min when challenged cells with TNFa (20, 100ng/ml) or IL-1b (20ng/ml). I could not detect the iNOS induction after stimulation with these cytokines after 6 hr, 16hr by qPCR.
Does anyone knows the reason and how I should change to get induction of iNOS ?
I use TNFa at 150 ng/ml and see NO production (looking at the protein via greiss assay) after 24h. Have you tried looking for NO? The assay is dead easy and only takes a couple of minutes! What kind of macrophages/microglia are you using?
Dear Charly
So far I checked only mRNA level of iNOS. Microglia is my first priority. I isolated primary microglia from postnatal brain of mice, the number of cells is not so many.
I think it will be difficult to detect the NO production because of the limited cell number. So that I try qPCR due to its higher sensitivity, but the fact that I could not detect changes in mRNA level.
Btw, what kinds of cell have you test?
Thank you for your comment !
In my case, I stimulated BV2 microglia cell line with LPS (100ng/ml) and detected iNOS (by Western Blot) and NO ( by greiss assay) after 20 hour. at this time point, I get clear WB signal as well as significant increase of NO production.
I also work on primary microglia, astrocyte and Raw264.7 macrophage as well. I think they are just different on the sensitive but not much.
you can try to incubate cell longer ( if the concentration of your stimulator is within middle range). I saw many of other paper they often try to measure iNOS and NO after 24 hours.
Good luck
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