Isolating Cell Populations from Brain Tissue Using Fluorescence-Activated Nuclei Sorting
Transcrição
Transfer a piece of fresh frozen human brain tissue to a suitable grinder containing cold lysis buffer.
Move the pestle up and down to mechanically break the tissue, releasing the cells.
The detergent molecules in the lysis buffer break open the cell membrane, releasing the nuclei.
Transfer the contents to a suitable centrifuge tube and add cold sucrose buffer to the bottom of the tube, creating a density gradient.
Centrifuge at high speed to separate the nuclei at the bottom of the tube.
Discard the supernatant and resuspend the nuclei in a buffered saline solution.
Add target-specific fluorophore-conjugated antibodies to the nuclei and incubate to allow the antibody to bind to its target nuclear protein.
Add nuclear staining dye to visualize the intact DNA.
Perform fluorescence-activated nuclei sorting to sort the nuclei of different cell populations based on distinct fluorescence signals.
