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.

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.

• Centrifuge Isolators - Devices used to separate components of a (e.g., cell) mixture.
• Sucrose Gradient Media - Medium used to separate components based on their density.
• Density Gradient Centrifugation - Method to separate cellular components using density.
• Density Gradient Medium - Medium in which components are separated in gradient centrifugation.
• Resident Immune Cells - Immune cells that permanently inhabit tissues (e.g., brain).

• Introduce Centrifuge Isolators - Tools used in separating mixed (e.g., cellular) components.
• Key Concepts - Principles of density gradient centrifugation (e.g., using density).
• Underlying Mechanisms - The nature of separation by density, e.g., gradient centrifugation.
• Connect to Experiment - Use of centrifugation to isolate resident immune cells from brain tissue.

• What are centrifuge isolators and how do they help in cellular separation?
• What is the concept behind density gradient centrifugation?
• What role does the density gradient medium play in the separation process?

• Practical Applications - Centrifugation used in scientific and medical labs (e.g., diagnostics).
• Industry Impact - Fields like neuroscience and immunology benefit (e.g., studying brain diseases).
• Societal Importance - Advances in understanding innate immunity (e.g., resident immune cells).
• Link to Scientific Advancements - Enhanced precision in cell separation techniques.