This article details a protocol for isolating microglia from transgenic zebrafish brain cell cultures. It highlights the use of fluorescent markers and specific antibodies to differentiate microglia from other cell types.
Under pathological conditions, activated microglia - specialized brain macrophages - help eliminate pathogens and cell debris. These microglia express unique receptors that differentiate them from other macrophages.
To isolate microglia, begin with a brain cell culture derived from transgenic zebrafish, expressing specific fluorescent molecules in different cell types.
The culture contains a mixture of neurons - expressing red fluorescence, macrophages - expressing green fluorescence, and non-fluorescent microglia.
Now, add a blocking reagent and incubate the culture with intermittent agitation. The blocking reagent binds to the non-specific sites on the cells and reduces the background signal.
Next, add primary antibodies and incubate. These antibodies bind to specific receptors expressed on the microglia surface.
Centrifuge the culture and remove the supernatant containing unbound antibodies. Resuspend the cell pellet in a fresh medium.
Subsequently, add fluorescent-dye conjugated secondary antibodies and incubate. These antibodies bind to the primary antibodies and impart a red color to the microglia-antibody complex.
Centrifuge the culture and remove unbound antibodies. Resuspend the pellet in a fresh medium.
Filter the cells to remove any debris.
Using a fluorescent activated cell sorter or FACS, sort the population of microglia cells based on their cell size and fluorescence.
The isolated microglia can be used for further applications.
To immunostain microglia, use 0.3 milliliters of Media A plus 2% NGS to resuspend the cell pellet. Then, split the cells between three 1.5-milliliter tubes: one for unstained cells to measure autofluorescence, one to measure the nonspecific binding of a secondary antibody to microglia, and a third as a test.
To all the tubes, add 1% Low Endotoxin, Azide-Free (LEAF) to block CD16/CD32 interactions with the Fc domain of immunoglobulins. Then, incubate the cells for 10 minutes with gentle agitation every 5 minutes.
Next, to the third tube, add the 4C4 antibody and incubate it for 30 minutes with gentle agitation every 10 minutes. Then, spin the tubes at 300 x g and 4 degrees Celsius for 10 minutes.
After discarding the supernatant, wash the pellet once with 0.5 milliliters of Media A plus 2% NGS before spinning the tubes again. Resuspend the cell pellet with 0.5 milliliters of Media A plus 2% NGS, then add 1% LEAF and incubate the cells for 10 minutes with gentle agitation every 5 minutes.
To tubes 2 and 3, add secondary antibodies and place the tubes in the dark. After spinning the tubes and discarding the supernatant, use 0.5 milliliters of Media A plus 2% NGS to wash the samples twice, then resuspend the cell pellets with 1 milliliter of Media A plus 2% NGS.
Run the cell suspension through a 35-micron cell strainer cap and transfer it into cold 5-millimeter FACS tubes on ice protected from light. Finally, carry out FACS sorting and RNA extraction according to the text protocol.
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