Isolating and Culturing Mouse Cerebral Pericytes
Transcription
Add buffer to the mouse brain tissue and homogenize it to release cells and microvessels.
Next, add the BSA-dextran solution to the homogenate. Mix and centrifuge to separate the components. Discard the supernatant.
Resuspend the microvessels in buffer, and pass them through a filter, removing larger vessels.
Now, centrifuge the filtrate and remove the supernatant. Resuspend the microvessels in buffer containing DNAses.
Add proteolytic enzymes to release endothelial cells and pericytes.
DNases degrade DNA.
Add buffer containing BSA to stop the enzyme activity. Centrifuge and discard the supernatant.
Resuspend the cells in media and plate them on a basement matrix-coated multi-well plate. Incubate, promoting cell adhesion.
Change media regularly.
Endothelial cells form a monolayer and support pericyte growth.
Subsequent cell passaging in pericyte media on a gelatin-coated multi-well plate promotes pericyte growth while eliminating endothelial cells.
