Mononuclear Cell Isolation from Mouse Brain: A Density Gradient Centrifugation Technique to Isolate Brain Resident Mononuclear Cells

Mononuclear cells in the brain, comprising lymphocytes and monocytes, play crucial roles in maintaining brain function and homeostasis.

To isolate these mononuclear cells, first, take a freshly harvested mouse brain perfused with saline. 

Perfusion allows removal of blood and its components from the blood vessels, enabling  isolation of brain-resident immune cells in the subsequent steps.

Now, rinse the brain with suitable media to remove adherent red blood cells. Follow up the rinse with mincing to acquire small tissue pieces.

Homogenize the tissue pieces to obtain a suspension of single cells and cell fragments.

To this suspension, then add chilled media and suitable density gradient medium in appropriate volumes to form a low-density gradient medium.

Next, add a specific volume of a high-density gradient medium; the high-density gradient media forms a separate layer at the bottom, creating a discontinuous density gradient.   

Centrifuge the suspension at high speed and low temperature conditions. Mononuclear cells occupy the interface between the two density gradient media layers.

Discard the top layer containing cellular debris and myelin - the fatty tissue sheath surrounding the nerve cell axons.

Transfer the interface layer into a fresh tube containing a suitable media and centrifuge.

Purified mononuclear cells sediment as pellet and can be resuspended in buffer for further analysis.