To begin, incubate a tube containing dissociated mouse hippocampal 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 five-milliliter microcentrifuge tube before centrifuging again. Subject the sample to Percoll gradient centrifugation, then resuspend the cell pellet in 100 microliters of MACS staining buffer, and incubate. Now, pipette one milliliter of MACS buffer to the sample before centrifuging.
Then, suspend the cell pellet in 500 microliters of MACS buffer. Next, rinse the positive selection columns of a magnetic separator with three milliliters of MACS buffer. Gently apply 500 microliters of the cell suspension onto a column.
After washing the columns three times with MACS buffer, place the columns on 15 milliliter conical tubes. Add five milliliters of MACS buffer onto the column. Next, centrifuge the samples at 300g for 10 minutes, then add one milliliter 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 500g 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 300g for 10 minutes before flow cytometric analysis. A positive PE fluorescence comparable to that obtained from synaptosomes at pH 4 was observed in CD11b-positive and CD45-positive microglia.
