Obtaining a Single-Cell Suspension from Mouse Hippocampal Tissue

Take perfused adult mouse hippocampi fragments in a dissociation tube containing papain and DNase.

Place the tube on a mechanical dissociator.

The dissociator mechanically disintegrates the tissue, loosening cells, including neurons and glial cells.

Papain breaks down the tissue's extracellular matrix, releasing cells. DNase degrades contaminating DNA.

Add buffer containing BSA, inactivating the enzymes.

Pass the suspension through a strainer to remove large debris.

Centrifuge the filtrate. Discard the enzyme-rich supernatant.

Resuspend the cells in buffer, and transfer them to a fresh tube.

Add density gradient media, mix, and overlay the mixture with buffer.

During centrifugation, cells and debris form separate layers based on their densities.

Remove the topmost buffer layer and the middle debris layer. 

Add buffer to the bottom layer containing the cells and mix. Centrifuge and discard the supernatant containing the density gradient media.

Resuspend the hippocampal cells in buffer for further analysis.