Hello!
We know of the traditional ways to isolate microglia from primary mixed cultures using shaking and tapping. However, these microglia are not very healthy and cause inconsistency in the reproducibility of experiments.
We are aware of methods to obtain primary astrocytes using clodronate or trichostatin-A. Is there a similar way to obtain pure microglia from a mixed glial population?
Obtaining pure microglia from a primary mixed culture can be challenging, as astrocytes and other cell types are often present. While it's not straightforward to completely remove astrocytes without affecting microglia, some methods can help enrich for microglial populations:Fluorescence-Activated Cell Sorting (FACS): Use cell surface markers specific to microglia (e.g., CD11b or CD45) combined with negative selection against astrocytic markers. FACS can separate different cell populations based on their surface markers.Magnetic-Activated Cell Sorting (MACS): Similar to FACS, MACS utilizes magnetic beads to separate cells based on specific surface markers. Microglial-specific antibodies can be used for positive selection, and astrocyte-specific antibodies for negative selection.Gradient Centrifugation: Density gradient centrifugation can be used to separate cells based on their density. This may help enrich microglia, but it might not completely eliminate astrocytes.Cell Culture Media Optimization: Adjusting the culture conditions can sometimes favor the survival or growth of microglia over other cell types. This may involve modifying the culture medium composition.
I would recommend looking into the Miltenyi kits for magnetic cell separation. If you have a mixed culture it is very easy to isolate highly pure populations of microglia (more than 95% on average) using the CD11b magnetic beads. The procedure is easy and viability is high. Generally microglia do not thrive on their own, so you want to use them quickly after generating the pure population and not keep them in culture for a long time. I have typically used pure microglia within 24-72h.
FACS also works if you have an instrument available.
Good luck!
Thanks a lot, Muhammad Umar & Eva Czirr for your helpful responses.
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