This protocol helps to isolate microglia from the microdissected demyelination lesions of the adult mouse brain to study the microglial characteristics in vivo. This technique saves time and has lower requirements for both experimenters and equipments. This method helps to isolate microglia from the animal's brain, especially those microdissected tissues in various disease states.
Begin by microdissecting the lesions labeled by neutral dye around the corpus callosum under a stereomicroscope. Centrifuge the dissected tissue at 300 times g for 30 seconds to collect the sample at the bottom of the tube. Preheat enzyme mix one and enzyme mix two to 37 degrees Celsius in an incubator.
Then, add 1, 950 microliters of preheated enzyme mix one to one sample, and digest in an incubator at 37 degrees Celsius for five minutes. Add 30 microliters of preheated enzyme mix two, and mix gently. After digestion, add four milliliters of cold PBS to the tube, and shake gently.
Centrifuge the tissue samples at 300 times g for 10 minutes at four degrees Celsius, and aspirate the supernatant slowly and completely. Resuspend the cell pellet gently with 1, 550 microliters of cold PBS. Add 450 microliters of the cold solution for debris removal, and mix them well.
Use a 1, 000-microliter pipette to overlay the mixture very slowly, and gently with two milliliters of cold PBS. Centrifuge the mixture, and then look for the three layers. Use a 1, 000-microliter pipette to completely aspirate the two top layers, and fill the tube up to five milliliters with cold loading buffer.
Gently invert the tube three times. Next, after centrifugation and aspiration of the supernatant, resuspend the cell pellet with 90 microliters of loading buffer, and add 10 microliters of CD11b beads. Mix well, and incubate for 15 minutes at four degrees Celsius.
After the incubation, add one milliliter of the loading buffer, and wash the cells by gently pipetting the liquid up and down with a 1, 000-microliter pipette. Centrifuge the cells at 300 times g for 10 minutes at four degrees Celsius, and aspirate the supernatant completely to remove the unbound beads. Resuspend the cells in 500 microliters of the loading buffer, and place the MS column with its separator for positive selection in the magnetic field.
Rinse the column with 500 microliters of the loading buffer to protect the cells and ensure the efficiency of magnetic sorting based on the manufacturer's protocol. Apply the cell suspension onto the MS column, and discard the flow-through containing the unlabeled cells. Add 500 microliters of the loading buffer to wash the column, and remove it from the separator.
Place the column on a 15-milliliter centrifuge tube, and add one milliliter of the loading buffer into the column. Finally, push the plunger to the bottom of the column to flush out the magnetically labeled cells. A schematic representation of the gating strategy used in flow cytometry analysis of microglia isolated from demyelination lesions of adult mice before and after the magnetic-activated cell sorting is shown here.
The graphical images represent the forward scatter height and side scatter height for selecting cells, forward scatter area and forward scatter height for selecting single cells, and CD11b-FITC and CD45-APC for selecting the myeloid cells and microglia. The proportion of microglia has increased significantly after the magnetic-activated cell sorting. The graphical image represents the gating strategy used in flow cytometry analysis for living and dead single cells after the magnetic-activated cell sorting.
Here, 7-aminoactinomycin D and side scatter height have been used to select the living cells. While attempting this procedure, remember to precisely microdissect the demyelinating lesions under a stereomicroscope based on the neutral red markings.
