Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Our research focus is on psychoneuroimmunology and we investigate the crosstalk of the immune system with the brain and the mind. I'm particularly interested in deciphering subgroups of patients based on their immune status to find tailored treatments for them. Recent developments in our field include, for instance, the development of PET ligands, but also include advanced technologies such as RNA sequencing, single-cell RNA sequencing, and spatial RNA sequencing to decipher immune-based therapies.

Our protocol addresses the challenge of quantifying microglial engulfment of synaptic material, which is crucial to understand their role in brain function as well as in disease pathology. Our protocol offers a fast and reliable quantification of engulfed synaptic material by microglia, and therefore provides insights into functional activity of microglia. Our protocol will inspire other research groups to include more functional assays in their research such as engulfment of synaptosomes, and this will lead a better and more comprehensive understanding of the role of microglia in neuroimmune interactions.

To begin, place a 70-micrometer strainer on a 5-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 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 in centrifuge.

Pipette 100 microliters of the staining master mix into the tube. Then incubate the samples for 30 minutes at 4 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 of macrophages.

To begin, incubate a tube containing dissociated mouse hippocampi tissue in a water bath at 37 degrees Celsius for 20 minutes. Transfer the cloudy cell suspension into a new 15-milliliter centrifuge tube. After discarding the supernatant, resuspend the cell pellet in five milliliters of the wash mix solution.

Next, pass the sample through a 70-micrometer filter into a 5-milliliter microcentrifuge tube before centrifuging again. Subject the sample to Percoll gradient centrifugation. Then resuspend the cell pellet in 100 microliters of max staining buffer and incubate.

Now, pipette one milliliter of max buffer to the sample before centrifuging. Then suspend the cell pellet in 500 microliters of max buffer. Next, rinse the positive selection columns of a magnetic separator with three milliliters of max buffer.

Gently apply 500 microliters of the cell suspension onto a column. After washing the columns three times with max buffer, place the columns on 15-milliliter conical tubes. Add five milliliters of max buffer onto the column.

Next, centrifuge the samples at 300 G for 10 minutes. Then add one milliliter of prewarmed DMEM to the pellet. Transfer 500 microliters of the final cell suspension into the wells of a 24-well plate.

Replace 250 microliters of each well with fresh prewarmed DMEM. Then add three micrograms of pHrodo Red-labeled synaptosomes over the media. Add the same volume of unlabeled synaptosomes to the negative controls.

After incubation, wash the wells with cold DPBS. Now pipette 200 microliters of trypsin-EDTA into each well. After a 35-second incubation, pipette one milliliter of DPBS containing FBS into each well.

Transfer the cell suspension into a five-milliliter polypropylene tube through a strainer. Then wash each well two times with 500 microliters of ice cold DPBS. Centrifuge collected samples at 500 G for five minutes.

Following this, incubate the cells in 100 microliters of the staining solution for 10 minutes on ice. Next, add CD11b and CD45 to the staining solution to make a final concentration of 1 to 100. Incubate the samples at four degrees Celsius for 20 minutes in the dark.

Pipette one milliliter of FACS buffer into the sample. Then subject it to a final centrifugation at 300 G for 10 minutes before flow cytometric analysis. A positive PE fluorescence comparable to that obtained from synaptosomes at pH four was observed in CD11b positive and CD45 positive microglia.