Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting
Transcription
Begin by microdissecting the lesions labeled by neutral dye around the corpus callosum under a stereomicroscope. Centrifuge the dissected tissue at 300 x g for 30 seconds to collect the sample at the bottom of the tube. Preheat enzyme mix 1 and enzyme mix 2 to 37 degrees Celsius in an incubator. Then, add 1,950 microliters of pre-heated enzyme mix 1 to one sample and digest in an incubator at 37 degrees Celcius for 5 minutes. Add 30 microliters of pre-heated enzyme mix 2, and mix gently.
After digestion, add 4 milliliters of cold PBS to the tube, and shake gently. Centrifuge the tissue samples at 300 x g for 10 minutes at 4 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 1000-microliter pipette to overlay the mixture very slowly and gently, with 2 milliliters of cold PBS. Centrifuge, the mixture and then look for the 3 layers. Use a 1000-microliter pipette to completely aspirate the 2 top layers, and fill the tube up to 5 milliliters with cold loading buffer. Gently invert the tube 3 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 4 degrees Celsius. After the incubation add 1 milliliter of the loading buffer, and wash the cells by gently pipetting the liquid up and down with a 1000-microliter pipette.
Centrifuge the cells at 300 x g for 10 minutes at 4:00 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 1 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.
