All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Single-Cell Suspension Preparation from Brain
<ol>
	<li>Dilute density gradient medium in 9:1 ratio with PBS in a 15 mL conical tube to yield a final 100% solution.</li>
	<li>Anesthetize mice at the peak of EAE with an intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg) and perfuse intracardially with 20 mL of sterile ice-cold PBS. Achieve this by slowly and steadily injecting PBS into the left ventricle of the heart using a 20 mL syringe and opening the right atrium.</li>
	<li>Cut the cranium carefully from the nose to the neck, then remove the brain from the cranial box into 10 mL of RPMI in 50 mL conical tubes. Mix well to remove adherent red blood cells. Then, remove the medium by aspiration and add 10 mL of RPMI.</li>
	<li>Place the brains and medium in a 100 mm dish. Finely chop with a razor blade.</li>
	<li>Transfer 6 mL from the dish to an ice-cold 7 mL sintered glass homogenizer with a clean pipette. Avoid leaving large quantities of tissue in the pipette. A small amount is unavoidable.</li>
	<li>Grind the brain using the "loose" plunger of the pestle first, then use the "tight" plunger until the suspension is homogeneous and pour into a prechilled 15 mL conical tube and keep on ice.</li>
	<li>After all the samples are homogenized, estimate the volume. Adjust the volume with RPMI to 7 mL. Then, place 3 mL of ice-cold 100% basement membrane matrix in a chilled 15 mL conical centrifuge tube and add 7 mL of the brain homogenate to yield a final 30% density gradient medium. Mix by inversion a couple of times. Do not vortex.</li>
	<li>To ensure a sharp interface, carefully and slowly add 1 mL of 70% underlay density gradient medium in RPMI with a 3 mL pipette.</li>
	<li>Centrifuge at 800 x g for only 20 min at 4 &#176;C. Set the acceleration to 1 and deceleration to 0. After centrifugation, aspirate almost all of the top phase, being careful to completely remove the myelin at the top (Figure 1).</li>
	<li>Remove the interface into a new 15 centrifuge tube. Adjust the volume to 10 mL with RPMI.</li>
	<li>Centrifuge at 500 x g for 10 min. After centrifugation, aspirate the supernatant. Resuspend the pellet in ~200 &#956;L of flow cytometry staining (FSC) buffer.</li>
</ol>

<table><tbody><tr><td>Dounce homogenizer</td><td>Wheaton</td><td>353107542</td><td></td></tr><tr><td>Percoll</td><td>GE</td><td>17-0891-01</td><td></td></tr></tbody></table>

mononuclear cell isolation from mouse brain: a density gradient centrifugation technique to isolate brain resident mononuclear cells

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

Source: Ji, Z. et al. <a href='https://www.jove.com/v/61050/flow-cytometric-analysis-lymphocyte-infiltration-central-nervous'>Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2020)
In this video, we perform the isolation of mononuclear cells from a mouse&#8217;s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

<img alt="Centrifugation diagram for myelin and mononuclear cell separation, 800xg, 20 min, 4&#176;C, density gradient." src="/files/ftp_upload/20621/20621_Figure1.jpg" data-altupdatedat="1753371239000" data-alt="Figure 1" /> 
Figure 1: Schematic of the Percoll gradient setup for isolation of mononuclear cells.

Source: Ji, Z. et al. Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis. J. ...

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.

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3.3K Views. 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 ...

Abstract

Protocol

<img title="figure-results-1" alt="Centrifugation diagram for myelin and mononuclear cell separation, 800xg, 20 min, 4&#176;C, density gradient." src="/files/ftp_upload/20621/20621_Figure1.jpg" data-altupdatedat="1753371239000" data-alt="Figure 1" /> 
Figure 1: Schematic of the Percoll gradient setup for isolation of mononuclear cells.

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

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Abstract

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List of materials used in this article
Name	Company	Catalog Number	Comments
Dounce homogenizer	Wheaton	353107542
Percoll	GE	17-0891-01