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

Cut the cranium carefully from the nose to the neck and transfer the brain of each animal from the cranial box into individual 50-milliliter tubes containing 10 milliliters of RPMI medium.

Mix the tubes well to remove the adherent red blood cells and remove the medium by aspiration. Add 10 milliliters of fresh medium to each tube and transfer the brains into individual 100-millimeter dishes.

Finely chop each brain with a razor and use a plastic pipette to transfer as much tissue from one dish at a time in 6 milliliters of medium into an ice-cold 7-milliliter sintered glass homogenizer.

Grind the brain using the "loose" plunger of the pestle before using the "tight" plunger to bring the tissue until the suspension is homogeneous. Then, pour the resulting tissue slurry into a prechilled 15-milliliter conical tube on ice.

When all the samples have been homogenized, adjust the volume in each tube to 7 milliliters with fresh medium before adding each tissue suspension to a new chilled 15-milliliter tube containing 3 milliliters of 100% basement membrane matrix per tube.

Mix by inversion a few times and use a 3-milliliter pipette to carefully and slowly add 1 milliliter of 70% density gradient solution under each tissue solution sample.

Separate the cells by density gradient centrifugation and remove almost all of the top layer, taking care to completely remove all of the myelin.

Transfer the interface into a new 15-milliliter tube and adjust the volume to 10 milliliters with fresh medium. Then, centrifuge the samples again and remove the supernatant before resuspending the pellets in about 100 microliters of medium per tube.