We acknowledge the contribution of Renata Sano in conceiving the initial protocol. A.d&#x2019;A. holds the Jewelers For Children (JFC) Endowed Chair in Genetics and Gene Therapy. This work was funded in part by NIH grants GM60905, DK52025 and CA021764, and the American Lebanese Syrian Associated Charities (ALSAC).

The following protocol is intended for the isolation and purification of MAMs and GEMs from mouse brain.
Solutions required for Isolation of mitochondria, MAMs and GEMs.
Fractionation to obtain crude mitochondrial preparation:
Solution A: 0.32 M Sucrose, 1 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2 + Protease Inhibitors (add fresh, as needed).
Solution B: 0.32 M Sucrose, 1 mM NaHCO3 + Protease Inhibitors (add fresh, as needed).
MAMs Isolation:
Isolation Medium: 250 mM Mannitol, 5 mM HEPES pH 7.4, 0.5 mM EGTA, 0.1% BSA.
Gradient Buffer: 225 mM Mannitol, 25 mM HEPES pH 7.5, 1 mM EGTA, 0.1% BSA (final concentration).
GEMs Isolation:
GEM Extraction Buffer: 25 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100 + Protease Inhibitors (add fresh, as needed).
GEM Solubilising Buffer: 50 mM Tris-HCl, pH 8.8, 5 mM EDTA , 1% SDS + Protease Inhibitors (add fresh, as needed).
1. First Step Subcellular Fractionation: Removal of Nuclei, Unlysed Cells and Cellular Debris
<ol>
 <li>Euthanize mouse in CO2 chamber.
 </li>
 <li>Immediately remove brain, halve, place in 2 ml tubes on ice and weigh. </li>
</ol>
Note: Keep brain halves separate throughout the entire procedure. The following applies to the treatment of a single brain half. 
<ol start="3">
 <li>Place half brain in a pre-chilled 2 ml glass Dounce tissue grinder containing 1 ml cold Solution A. Homogenize with 15 total strokes of a large clearance pestle. </li>
 <li>Transfer to a 15 ml falcon tube, on ice, and dilute homogenates up to 10 volumes w/v, e.g. 0.225 g to 2.25 ml. </li>
 <li>Centrifuge sample at 1,400 x g for 10 min at 4 &deg;C.
 </li>
 <li>Carefully remove the supernatant and transfer to a 30 ml round-bottom glass centrifuge tube, on ice. Make sure not to disturb the pellet. </li>
 <li>Resuspend the pellet in the same 10 volumes of Solution A. Homogenize in the same grinder, with 3-6 strokes of a small clearance pestle, 1 ml at a time. </li>
 <li>Transfer to a fresh 15 ml falcon tube and centrifuge at 710 x g for 10 min at 4 &deg;C. This results in a pellet of nuclei and cell debris. </li>
 <li>Carefully remove the supernatant and pool with the supernatant saved in step 1.6. </li>
</ol>
2. Second Step: Crude Mitochondria Isolation
<ol>
 <li>Centrifuge supernatants at 13,800 x g for 10 min at 4 &deg;C. </li>
 <li>Transfer supernatant to an Ultra-Clear Beckman Centrifuge Tube, on ice </li>
 <li>Resuspend pellet in 10 volumes Solution A. Homogenize in the same grinder, with 3-6 strokes of a small clearance pestle, 1 ml at a time. </li>
 <li>Centrifuge as step 2.1. </li>
 <li>Repeat steps 2.2-2.4. </li>
 <li>The resulting pellet is an enriched mitochondrial fraction. Pool the supernatants (cytosol and ER), cover with parafilm and keep on ice.</li>
 <li>Resuspend the pellet with 6 strokes of a small clearance pestle, in 4.8 ml/g (g is referred to the original brain weight) of Solution B, in a fresh homogeniser. </li>
 <li>Using glass Pasteur pipettes, prepare a Discontinuous Sucrose Gradient in an Ultra-Clear Beckman Centrifuge Tube, adding subsequently to the bottom of the tube as follows, carefully, ensuring no bubbles are formed: 
 Resupended Pellet (0.32 M Sucrose) 
 3 ml 850 mM Sucrose (in 1 mM NaHCO3) 
 3 ml 1 M Sucrose (in 1 mM NaHCO3) 
 3 ml 1.2 M Sucrose (in 1 mM NaHCO3) </li>
 <li>Centrifuge at 82,500 x g for 2 hr at 4 &deg;C. The resulting separation at the end of the gradient will produce three bands and a pellet: 1) myelin and other membrane contaminants (0.32 M - 0.85 M interface); 2) ER, Golgi, plasma membranes (0.85 M - 1 M interface); 3) synaptosomes (1 M - 1.2 M interface); 4) crude mitochondria (pellet) that is used for the subsequent purification steps
 
 </li>
</ol>
3. Isolation of Mitochondria-associated ER Membrane, MAMs
<ol>
 <li>Resuspend the freshly isolated crude mitochondria from one brain half, in 2 ml Isolation Medium containing freshly added protease inhibitors. 
 </li>
 <li>Prepare a 30% Percoll gradient with Gradient Buffer 8 ml per half brain, and place in an Ultra-Clear Beckman Centrifuge Tube. </li>
</ol>
* Note: Make gradient buffer more concentrated (1.43 times) to achieve correct final concentration after diluting Percoll to 30%.
<ol start="3">
 <li>Layer mitochondrial suspension on top of prepared gradient SLOWLY to avoid any bubbles.</li>
 <li>Centrifuge at 95,000 x g for 30 min, 4 &deg;C.
 </li>
 <li><ol class="lroman">
 <li> Remove the Heavy Fraction (lower band) FIRST with a glass Pasteur pipette and transfer to a fresh round-bottom glass tube, on ice.</li>
 <li> Remove the Light Fraction (upper band) SECOND with another glass Pasteur pipette and transfer to a separate fresh round-bottom glass tube, on ice. </li>
 </ol>
 </li>
 <li>Dilute both fractions collected in step 3.5 with 10 ml Isolation Medium and centrifuge at 6,300 x g for 10 min at 4 &deg;C.
 </li>
 <li>Discard the supernatant from the Heavy Fraction obtained in step 3.6, resuspend the pellet with another 10 ml Isolation Medium and centrifuge again, as step 3.6. The resulting pellet will be the pure mitochondrial fraction. </li>
 <li>Transfer the supernatant from the Light Fraction obtained in step 3.6 to an Ultra-Clear Beckman Centrifuge Tube, on ice. Discard the pellet. </li>
 <li>Add sufficient Isolation Medium to the supernatant obtained in step 3.8 to fill the tube and centrifuge at 100,000 x g for 1 hr, 4 &deg;C. The resulting pellet will be the MAMs fraction. Carefully remove and discard the supernatant. </li>
</ol>
Note: At this step, you may also centrifuge at 100,000 x g for 1 hr, 4 &deg;C the supernatant saved at step 2.6. The pellet will be the ER fraction and supernatant the Cytosolic fraction.
4. Extraction of Glycosphingolipid-enriched Microdomains, GEMs 
<ol>
 <li>Lyse pure mitochondria and/or MAM fractions in 500 &mu;l-1 ml of Extraction Buffer for 20 min on ice.</li>
 <li>Centrifuge lysates at 15,300 x g for 2 min at 4 &deg;C. </li>
 <li>Collect supernatants (Triton X-100 soluble material) and re-centrifuge pellets for 2 min to remove remaining soluble material. (Triton X-100 extracted Mitochondria and/or Triton X-100 extracted MAMs).</li>
 <li>Solubilize pellets in Solubilising Buffer. This solubilized material represents the GEM fractions and can be used for further analysis.</li>
</ol>

<ol>
	<li>Poburko, D., Kuo, K. H., Dai, J., Lee, C. H., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+junctions+promote+targeted+Ca2++signaling+in+smooth+muscle:+why+two+membranes+are+better+than+one.">Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one.</a> Trends Pharmacol. Sci. 25 (1), 8-15 (2004).</li><li>Pani, B., Ong, H. L., Liu, X., Rauser, K., Ambudkar, I. S., Singh, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+determine+clustering+of+STIM1+in+endoplasmic+reticulum-plasma+membrane+junctions+and+regulation+of+store-operated+Ca2++entry+(SOCE.">Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE.</a> J. Biol. Chem. 283 (25), 17333-17340 (2008).</li><li>Levine, T., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum:+one+continuous+network+compartmentalized+by+extrinsic+cues.">Endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues.</a> Curr. Opin. Cell Biol. 17 (4), 362-368 (2005).</li><li>Sano, R., Annunziata, I., Patterson, A., Moshiach, S., Gomero, E., Opferman, J., Forte, M., d'Azzo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1-ganglioside+accumulation+at+the+mitochondria-associated+ER+membranes+links+ER+stress+to+Ca(2+)-dependent+mitochondrial+apoptosis.">GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.</a> Mol. Cell. 36 (3), 500-511 (2009).</li><li>Raturi, A., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+the+endoplasmic+reticulum+and+the+mitochondrion+tie+the+knot:+The+mitochondria-associated+membrane+(MAM).">Where the endoplasmic reticulum and the mitochondrion tie the knot: The mitochondria-associated membrane (MAM).</a> Biochim. Biophys. Acta. , (2012).</li><li>Giorgi, C., De Stefani, D., Bononi, A., Rizzuto, R., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+link+between+the+mitochondrial+network+and+the+endoplasmic+reticulum.">Structural and functional link between the mitochondrial network and the endoplasmic reticulum.</a> Int. J. Biochem. Cell Biol. 41 (10), 1817-1827 (2009).</li><li>d'Azzo, A., Tessitore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sano+Gangliosides+as+apoptotic+signals+in+ER+stress+response.">Sano Gangliosides as apoptotic signals in ER stress response.</a> Cell Death Differ. 13, 404-414 (2006).</li><li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+permeabilization+in+cell+death.">Mitochondrial membrane permeabilization in cell death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Rizzuto, R., Brini, M., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdomains+with+high+Ca2++close+to+IP3-sensitive+channels+that+are+sensed+by+neighboring+mitochondria.">Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.</a> Science. 262, 744-747 (1993).</li><li>Lynes, E. M., Bui, M., Yap, M. C., Benson, M. D., Schneider, B., Ellgaard, L., Berthiaume, L. G., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylated+TMX+and+calnexin+target+to+the+mitochondria-associated+membrane.">Palmitoylated TMX and calnexin target to the mitochondria-associated membrane.</a> EMBO J. 31 (2), 457-470 (2011).</li><li>Fujimoto, M., Hayashi, T., Su, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cholesterol+in+the+association+of+endoplasmic+reticulum+membranes+with+mitochondria.">The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria.</a> Biochem. Biophys. Res. Commun. 417 (1), 635-639 (2012).</li><li>Grimm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ER-mitochondria+interface:+the+social+network+of+cell+death.">The ER-mitochondria interface: the social network of cell death.</a> Biochim. Biophys. Acta. 1823 (2), 327-334 (2012).</li><li>Vance, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+synthesis+in+a+membrane+fraction+associated+with+mitochondria.">Phospholipid synthesis in a membrane fraction associated with mitochondria.</a> J. Biol. Chem. 265, 7248-7256 (1990).</li><li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells.">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat. Protoc. 4 (11), 1582-1590 (2009).</li><li>Pizzo, P., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+lipid+rafts:+structure+and+function.">Lymphocyte lipid rafts: structure and function.</a> Curr. Opin. Immunol. 15 (3), 255-260 (2003).</li></ol>

None of the authors have any conflicts of interest to declare.

The sites of contact between intracellular membranes or between organelles and the plasma membrane of cells represent dynamic signaling platforms for basic cellular processes. The accurate characterization of their function and composition under both physiological and pathological conditions requires reliable and reproducible purification protocols. The methods detailed here have been specifically optimized by our lab for the isolation and purification of the MAMs and their corresponding GEMs from the adult mouse brain. ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Fractionation</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Fisher Scientific</td>
 <td>S5-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate</td>
 <td>Sigma-Aldrich</td>
 <td>S-5761</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride, Hexahydrate</td>
 <td>Fisher Scientific</td>
 <td>BP214-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride, Dihydrate</td>
 <td>Sigma-Aldrich</td>
 <td>C-5080</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>MAM </td>
 </tr>
 <tr>
 <td>D-Mannitol</td>
 <td>Sigma-Aldrich</td>
 <td>M9546-250G</td>
 <td></td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>Fisher Scientific</td>
 <td>BP310-500</td>
 <td></td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378-250G</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA, Fraction V, Heat Shock, Lyophilizate</td>
 <td>Roche</td>
 <td>03-116-964-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Percoll</td>
 <td>GE</td>
 <td>17-0891-02</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>GEM </td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T9284-500 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Roche</td>
 <td>03-118-142-001</td>
 <td></td>
 </tr>
 <tr>
 <td>HCl</td>
 <td>Fisher Scientific</td>
 <td>A144S-500</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher Scientific</td>
 <td>BP120-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate (SDS)</td>
 <td>Fisher Scientific</td>
 <td>BP166-500</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Common </td>
 </tr>
 <tr>
 <td>Protease Inhibitors Tablets, Complete EDTA-free</td>
 <td>Roche</td>
 <td>11-873-580-001</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>2 ml Douce All-Glass Tissue Grinders</td>
 <td>Kimble Chase</td>
 <td>885300-0002</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml Polypropylene Conical Centrifuge Tubes, BD Falcon</td>
 <td>BD</td>
 <td>352097</td>
 <td></td>
 </tr>
 <tr>
 <td>30 ml Round-Bottom Glass Centrifuge Tubes</td>
 <td>Kimble Chase</td>
 <td>45500-30</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml Round-Bottom Glass Centrifuge Tubes</td>
 <td>Kimble Chase</td>
 <td>45500-15</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge tubes, Ultra-Clear, Thinwall, 14x89 mm</td>
 <td>Beckman-Coulter</td>
 <td>344059</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Cole-Parmer</td>
 <td>PM996</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable Borosilicate Glass Pasteur Pipets, 9"</td>
 <td>Fisher Scientific</td>
 <td>13-678-20C</td>
 <td></td>
 </tr>
 </tbody>
</table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Based on our experience with using this protocol we can safely recommend it for the isolation and purification of MAMs, GEMs and mitochondrial fractions from mouse brain. The procedure as outlined is highly reproducible and consistent. In Figure 1 we show a representative image of the way pure mitochondria and MAMs layer on a Percoll gradient (step 3.4). A defined, milky band containing purified mitochondria (Mito-P) segregates at the bottom of the ultracentrifuge tube, while the MAM fraction makes up a ...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Research

JoVE Journal

Neuroscience

24.2K Views.  St Jude Children's Research Hospital. This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Video: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Studying Pre-formed Fibril Induced &amp;#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Mouse Brain Dissection for Microglia Isolation

Begin by placing the isolated mouse brain tissue in 1 milliliter of digestion buffer per brain in separate Petri dishes on ice. Thoroughly chop the brain tissues into small pieces using a clean scalpel blade. Using a plastic transfer pipette with its tip cutoff, carefully transfer minced brains into individual wells inside a 24-well plate on ice.Cover the plate with a large piece of transparent, flexible film, close the lid, and incubate on ice for 30 minutes. Next, transfer the digested brain solution into individual 7-milliliter iced glass dounce homogenizers on ice containing 5 milliliters of cold FACS buffer. Gently dounce each brain with a loose pestle until a single-cell suspension is obtained.Softly dounce with the tight pestle three to four times to ensure a single-cell suspension before transferring to 15-milliliter polypropylene tubes.

Mouse Brain Immune-Enriched Fragment Isolation for Histone PTM Quantification

Begin by adding 2.125 milliliters of isotonic density gradient to a 15 milliliter polypropylene tube containing murine brain homogenate. Top up each tube with fax buffer to a final volume of 8.5 milliliters. Gently invert the tubes 20 times to mix thoroughly.Using a narrow graduated transfer pipette, gently underlay four milliliters of pink, 37%density gradient establishing two clean layers. Switch transfer pipettes and underlay two milliliters of the blue, 70%density gradient below the 37%layer. Transfer the tubes to a centrifuge cool to four degrees Celsius and spin them at 500 G for 20 minutes with the braking ramp set to the lowest setting.After centrifugation, discard the myelin from the top of the 15 milliliter tube using a clean transfer pipette. Carefully collect the top fragment of the density gradient into a clean 15 milliliter polypropylene tube using another transfer pipette. Next, gather all cells in the sample by slowly circling the pipette along the sides of the tube while collecting the immune enriched fragment.Transfer the immune enriched fragment into a new 15 milliliter polypropylene tube. Wash the sample by adding 10 milliliters of fax to the tube. Gently invert the tube 20 times to mix thoroughly.Spin the tubes in a cooled centrifuge with the braking ramp set to the lowest setting to pellet the isolated immune cells.