Preparing a Single-Cell Suspension of Immune Cells from Murine Brain Tissue

Begin with a murine brain with neural, non-neural, and immune cells within the brain's extracellular matrix or ECM.

Mince the tissue and resuspend it in a culture medium.

Transfer these tissue fragments to a tube and treat them with collagenase and DNase-I enzymes.

Collagenase digests the ECM, releasing individual cells, while DNase-I degrades free DNA to prevent cell clumping.

Then, add EDTA to inactivate the collagenase enzymes.

Add a culture medium. Centrifuge and remove the supernatant.

Add a density gradient solution and the culture medium to the cell pellet, creating a low-density gradient layer.

Mix and then underlay the solution with a high-density gradient solution to establish a density difference.

Centrifuge at low speed to separate immune cells at the interface, with lighter cell debris at the top and heavier neuronal and glial cells at the bottom.

Remove the upper layer, and collect the immune cells, and transfer them to another tube.

Centrifuge and remove the supernatant, then resuspend the cells in a medium, forming a single-cell suspension for downstream applications.