To begin, place a 70 micrometer strainer on a five milliliter polypropylene tube. Pipette 500 microliters of the neural cell medium over it. Transfer the dissociated mouse hippocampi tissue homogenate into the tube through the strainer.
Then pipette one milliliter of cold neural cell medium over the homogenizer twice to rinse it. Now, add 500 microliters of ice cold DPBS to the pellet to resuspend it. Make up the volume of the suspension to 1.5 milliliters with DPBS.
Pipette 500 microliters of isotonic percoll solution to the sample. Overlay the solution with two milliliters of cold DPBS in centrifuge. Then aspirate the top layer and the myelin disc in the middle phase.
Next, add four milliliters of cold DPBS into the tube, aspirate the supernatant after centrifuging it. Then resuspend the cells in 100 microliters of fixable viability staining solution. Pipette one milliliter of cold DPBS to the sample before centrifuging the sample at 300 x g for five minutes.
After aspirating the supernatant, add 100 microliters of CD16, CD32 staining solution. Vortex the sample for five seconds, then incubate. After incubation, add one milliliter of FACS buffer into the sample and centrifuge.
Pipette 100 microliters of the staining master mix into the tube. Then incubate the samples for 30 minutes at four degrees Celsius in the dark. Now, add one milliliter of FACS buffer to the sample before centrifuging as before.
Resuspend the pellet in 250 microliters of fixation buffer after discarding the supernatant. Next, add two milliliters of permeabilization buffer to the tube and centrifuge again. Pipette 100 microliters of vGLUT1 staining solution to the pellet.
Then vortex the mixture for about five seconds before incubation and centrifugation. Resuspend the cell pellet in 250 microliters of FACS buffer. Finally, filter the samples through a 40 micrometer filter.
A higher vGLUT1-PE fluorescent signal was observed from the hippocampal microglia compared to the isotype control. A higher vGLUT1-PE fluorescent signal was found in the microglia from the olfactory bulb and a lower signal in the cerebellum compared to the hippocampus. The lowest signal intensity was detected in the spleen macrophages.
