Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

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.

For tissue homogenization, add 3 milliliters of prechilled HBSS to the prechilled glass mortar of a Dounce tissue grinder, and transfer the half of one of the harvested brain to the mortar. Gently macerate the tissue with 10 strokes of pestle A, followed by 10 strokes of pestle B, and transfer the homogenized mixture into a new 15-milliliter conical tube.

Fill the tube to a final volume of 10 milliliters with prechilled HBSS for centrifugation and resuspend the pellet in 7 milliliters of fresh HBSS with vortexing before holding the sample on ice. For debris removal, add 3 milliliters of prechilled isotonic solution to each digested or homogenized sample, and gently vortex the samples to make sure they are homogeneously mixed.

Next, centrifuge the samples and carefully remove the whitish disk of debris and myelin floating at the surface of the solution. When the debris has been discarded, collect all but the last 100 microliters of the supernatant from each tube without disturbing the pellets. Resuspend each pellet in 1 milliliter of FACS/BL solution for transfer into individual 1.5-milliliter microcentrifuge tubes.

After the centrifugation with the particle solution, it's important to remove the debris disk and the supernatant very carefully, without dislodging the cell pellet, to avoid sample loss.

After centrifugation, carefully aspirate the supernatant from each tube and resuspend the pellets in 350 microliters of fresh FACS/BL per tube.