eoe sub articles

EoE Sub Articles

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
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	<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...

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="Figure 1" src="/files/ftp_upload/20621/20621_Figure1.jpg" /> 
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

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.&#160;
Perfusion allows removal of blood and its components from the blood vessels, enabling&#160; 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.&#160;&#160;&#160;
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.

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

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