I am currently learning to culture primary microglia cells from P0-P2-day-old pups, and after 14 days of culturing, I got these cells. I performed IBA1 staining to check purity, but still, I am worried since when I performed the seahorse experiment, irrespective of the cell seeding density, I was getting very low signals.
Hi Soniya Charles
How were the microglia isolated from the astrocytes? Was it through rigorous shaking? Or you used a commercial kit?
Can you share a high magnification image? To check the morphology of cells.
Thanks,
On day 14, T75 flask was shaken for 3 hours at 37°C and 250 rpm, collected the supernatant, and centrifuged for 10 minutes at 250 rpm.
Since you mentioned that these are separated from mixed glia cultures, then I would say they look fine. How long were they in this separated culture? They generally look like this after a day. Sometimes even after overnight incubation. As mentioned above, a higher magnification image would be helpful, but they seem fine.
Yes, these are on the day after harvest.
Iba1+ DAPI stained microglia picture under 20x and 43x is attached herewith for your reference.
They definitely look like how they are supposed to. How many brains do you plate in a T75 flask.? We generally plate 3 brains per flask, and if the culture is really good, we get around 2.5mil microglia per flask.
2 brains per flask. 25 ml media.
How much ml media are you adding for 3 brains?
Coated flask? If so, which coating is highly recommended for primary microglia?
I have too many things to clarify. Can you please share your working protocol?