I am working with SIM-A9 murine microglia. According to the ATCC and several publications, these cells can be stimulated by LPS at a dose as low as 2.5 ng/mL.
In the first round of experiments, only a combination of LPS 10 ng/mL and IFN 10 IU/mL yielded a coloration. However, for some reason, I can't replicate these results. I have tried many things to no avail.
- I tried cell dissociation by scraping and by an EDTA/glucose-based dissociation solution.
- I tried complete and serum-free media
- I tried different doses of LPS (2.5 ng/mL all the way up to 2 ug/mL)
- I even tried different seeding densities and different incubation times.
No matter what I do, I can't get the assay to work. Does anyone have any idea as to what might be the problem and how to solve it?
Thanks
Hey Mohamed,
I can only share with you our expertise with peritoneal macrophages and bone-marrow dervied macrophages.
What we do is the following:
1. Seed your your microglia in full medium plus serum in a 96er flat bottom plate (serum does not influence the assay) in technical triplicates.
You need:
a) untreated cells
b) +IFNg (10 ng/ml f.c.)
c) +LPS (5 µg/ml f.c.)
d) +IFNg+LPS
e) Only medium without cells for background
2. After seeding and before you add the substances, let them rest for 2h.
3. Add IFNg to your wells with 10 µl (110 µl total in wells) and incubate for 24h.
4.The next day add LPS to your wells with 10 µl (120 µl total) and incubate for another 24h, so your IFNg incubates 48h and your LPS 24h in total.
5. After the incubation, take 50µl from each well and transfer it to a new 96er clear flat bottom well plate.
6. Add 50 µl Griess-Reagent 1 (1 % Sulfanilamid in 2.5% H3PO4 buffer) and incubate 10 mins in the dark.
7. Add 50 µl Griess Reagent 2 (0.1 % Naphtylethylendiamindihydrochloride in 2.5% H3PO4 buffer) and incubate 10 mins in the dark.
8. Read absorption at 550 nm.
I hope that helps you,
All the best and stay healthy,
Marc
Marc Herb Thank you so much Marc
This is a very different protocol from the one I am currently doing.
I will give it a try. Thanks again.
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