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. Brain dissociation and magnetic microglial isolation
NOTE: All cell manipulations and resuspensions must be performed with a 1,000 &#181;L pipette with great caution. Applying a high mechanical action may activate or kill microglia cells.
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	<li>Transfer 12 brain pieces (~1.2 g) per...

magnetic isolation of microglia from mouse pup brains

Source: Bokobza, C. et al., <a href="https://app.jove.com/v/62964">Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures.</a> J. Vis. Exp. (2022)
In this video, we describe a protocol for isolating CD11b cells, including microglia, from mouse pup brains using magnetic-activated cell sorting. The brain tissues are homogenized to obtain single-cell suspensions, which are incubated with anti-CD11b antibody-coated microbeads, followed by a magnetic column-based separation to purify the CD11b+ cell population.

<img alt="Figure 1" src="/files/ftp_upload/21576/21576_Figure1.jpg" /> 
Figure 1: Schematic representation of brain dissociations and microglial cells&#39; isolation. (A-C) After P8 mouse brain dissection and removing olfactive bulbs and cerebellum, brains were first transferred to a Petri dish with HBSS+/+, and then to dissociation tubes containing the dissociation mixture. The C-tubes were placed on the dissociator (with the heating mode), and the NTDK program was started (<strong...

Magnetic Isolation of Microglia from Mouse Pup Brains

Source: Bokobza, C. et al., Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (2022)
In this video, we ...

Microglia &#8212; the brain-resident macrophages &#8212; express CD11b, a cell-surface integrin.
To isolate microglia from mouse pup brains, take cerebrum fragments in dissociation tubes containing buffer with papain, a proteolytic enzyme, and deoxyribonuclease.
Place the tubes on a mechanical dissociator. During the run, the dissociator mechanically disrupts the tissue.
Papain digests the tissue&#8217;s extracellular matrix, releasing cells, including microglia, into suspension. Deoxyribonuclease degrades free DNA in suspension, preventing inefficient cell isolation.
Centrifuge. Complete the mechanical dissociation by pipetting. Transfer the suspension through a cell strainer to remove debris and cell clumps, and obtain a homogeneous single-cell population.
Incubate with superparamagnetic microbeads functionalized with anti-CD11b antibodies. The microbeads specifically bind to CD11b on cells, including microglia.
Post-incubation, centrifuge. Remove the supernatant containing unbound beads. Resuspend the cells in buffer. Load onto a column placed on a magnetic separator. The column comprises a matrix with ferromagnetic beads.&#160;
When placed on the separator, the spheres within the column amplify the magnetic field, retaining the microbead-bound CD11b+ cells within the column, while other cells flow through.
Remove from the separator and use buffer to elute the CD11b+ cells from the column. Centrifuge the suspension and resuspend the CD11b+ cells in a microglia medium.
The isolated cells are ready for further analysis.

Magnetic Isolation of Microglia from Mouse Pup Brains

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