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Inspecting microglia dissected out of neonatal brains

Today in the lab I was looking at brain microglia which were isolated from a mixed culture of both microglia and astrocytes three days earlier. I had previously dissected these cells out of neonatal brains and grew them in a flask. To look at them today, I used a phase contrast microscope. I was looking at the morphology of the microglia, as well as the purity of the culture, to ensure my isolation experiment was successful. The image below shows the cells I was looking at today.

Microglia grown in culture, isolated from a mixed glia culture of cells that were dissected out of neonatal brains.
Microglia grown in culture, isolated from a mixed glia culture of cells that were dissected out of neonatal brains.

Detailed methods of today’s experiment

Microglia growing in culture were isolated using a mild trypsinization method. Briefly, the culture medium was removed, and the cells were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS). The mixed glia-conditioned medium was passed through a 0.2 μm filter to remove any cell debris. The cells received trypsin-EDTA diluted 1:4 in Dulbecco’s Modified Eagle’s Medium (DMEM) and incubated for 45 minutes at 37°C, 5% CO2 to detach the astrocyte layer, followed by culture media to inactivate trypsin activity. All the medium was then aspirated to remove the detached astrocytes and oligodendrocytes, and the cells were washed with DPBS. The remaining cells were the attached microglia which were kept in 50% mixed glia-conditioned culture medium at 37°C, 5% CO2 for two days until they became ramified again.