Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization
Transcription
Begin by placing the tissue grinder components on ice. This device includes a mortar and two pestles of different diameters.
Pour a chilled salt buffer into the mortar and then add mouse brain tissue.
The buffer maintains osmotic balance, preserving cell structure and function.
Grind the tissue with multiple gentle strokes using the smaller diameter pestle, which mechanically shears the tissue into smaller pieces.
Next, stroke with the larger diameter pestle, facilitating further breakdown of tissue into its component cells.
Transfer the mixture to a tube and centrifuge.
Remove the supernatant.
Add chilled salt buffer, and vortex to break the cell clumps and the myelin.
Next, add a density gradient medium and centrifuge.
Due to differences in shape and mass, cells will settle in the lower layer, while debris and myelin accumulate in the top layer.
Discard the supernatant.
Add a suitable solution to obtain a multiple-cell suspension for further use.
