Isolating Cell Populations from Brain Tissue Using Fluorescence-Activated Nuclei Sorting
Transcrição
Begin dissecting approximately 200 to 400 milligrams of tissue from a fresh, frozen human adult cortex tissue sample. Place the tissue in a 7-milliliter glass tissue Douncer containing 4 milliliters of ice-cold lysis buffer on ice, and grind the tissue approximately 50 times.
Transfer the homogenate to a 12-milliliter ultracentrifuge polypropylene tube. Use a 5-milliliter pipette to add 6.5 milliliters of ice-cold sucrose buffer to the bottom of the ultracentrifuge tube without disturbing the interface between the sucrose and the tissue homogenate.
To collect the nuclei, ultracentrifuge the lysate for one hour at 101,814 times g, and carefully aspirate the supernatant and debris without disturbing the pellet. Add PBS to the ultracentrifuge tube, resuspending the nuclei pellet.
For fluorescence-activated sorting of the isolated nuclei, add approximately 20 microliters of the resuspended sample to an antibody solution containing AlexaFlour555-conjugated mouse anti-NeuN antibody. Add approximately 20 microliters of the resuspended sample to an antibody solution containing APC-conjugated mouse anti-PAX6 antibody. After a one-hour incubation, at 4 degrees Celsius with shaking protected from light, add DAPI at a 1 to 1,000 ratio to all of the samples and controls.
