Figures were created using BioRender. Research is funded by Inserm, Universit&#233; de Paris, Horizon 2020 (PREMSTEM-874721), Fondation de France, Fondation ARSEP, Fondation pour la Recherche sur le Cerveau, Fondation Grace de Monaco, and an additional grant from Investissement d&#39;Avenir -ANR-11-INBS-0011-NeurATRIS and Investissement d&#39;Avenir -ANR-17-EURE-001-EUR G.E.N.E.

The protocol was approved, and all the animals were handled according to the institutional guidelines of Institut National de la Sante&#769; et de la Recherche Scientifique (Inserm, France). Magnetic isolation of microglia from the brains of 24 OF1 mouse pups (both male and female) at P8, divided into 6-well, 12-well, or 96-well plates, are presented. The experimental work was performed under a hood to maintain sterile conditions.
1. Preparation of sterile solutions for isolation and cell culture
<ol>
	<li>Prepare 50 mL of 1x Hanks&#39; Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (HBSS-/-) from the commercially available 10x solution (see Table of Materials).</li>
	<li>Prepare dissociation mixture according to the composition provided in Table 1 using a commercially available tissue dissociation kit (see Table of Materials).</li>
	<li>Prepare 200 mL of 1x HBSS with Ca2+ and Mg2+ (HBSS+/+) from the commercially available 10x solution.</li>
	<li>Prepare 200 mL of 1x PBS + 0.5% BSA (referred to as cell sorting buffer).</li>
	<li>Prepare CD11b-microbeads according to Table 2.</li>
	<li>Prepare 500 mL of macrophage serum-free culture medium (SFM) + 1% of Penicillin-Streptomycin (P/S). Make aliquots of 50 mL tubes and store them at 4 &#176;C. This is referred to as the microglia medium later in the text. 
	NOTE: All isolation solutions must be freshly prepared under sterile conditions on experimentation day and kept on ice. Microglia cell culture medium can be prepared, aliquoted in 50 mL tubes, and kept at 4 &#176;C for future use. Filtration is not needed.</li>
</ol>
2. Brain dissection
<ol>
	<li>Decapitate the pup&#39;s head using large scissors without previous general anesthesia.</li>
	<li>Cut the skin from the neck to the nose following the sagittal suture (15-20 mm) with small scissors (Figure 1A-C).</li>
	<li>Insert a small scissor&#39;s tips within the Foramen magnum parallel to the skull. Cut from each side carefully to the eyes (Figure 1D,E).</li>
	<li>Cut between the eyes with small scissors to detach the skull and brain from the head (Figure 1F).</li>
	<li>With two forceps, grab the skull close to the olfactory bulbs and tear the skull carefully, taking care not to damage the underlying brain (Figure 1G-I).</li>
	<li>Remove cerebellum and olfactive bulb with a razor blade and cut the brain into two pieces (Figure 1J).</li>
	<li>Place the brain pieces in a Petri dish containing 30-40 mL of HBSS-/- (Figure 1K).</li>
</ol>
3. 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.
<ol>
	<li>Transfer 12 brain pieces (~1.2 g) per dissociation tube containing dissociation mixture according to Table 1. For 24 pups, four C-Tubes were needed (Figure 2A-B).</li>
	<li>Place C-Tubes on the dissociator (with the heating mode). Start the optimized NTDK program in the equipment according to the manufacturer&#39;s instruction (Figure 2D).</li>
	<li>Centrifuge at 300 x g for 20 s (at 4 &#176;C) to collect all the cells. Complete the mechanical dissociation by pipetting three times up and down with a 1,000 &#181;L pipette (Figure 2E).</li>
	<li>Transfer the cells to four 15 mL tubes + strainers. Rinse the strainers with 10 mL of HBSS+/+ (Figure 2F).</li>
	<li>Centrifuge at 300 x g for 10 min&#160;(at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of HBSS+/+ (Figure 2G).</li>
	<li>Centrifuge at 300 x g for 10 min&#160;(at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 6 mL of sorting buffer (step 1.4) (Figure 2H).</li>
	<li>Centrifuge at 300 x g for 10 min&#160;(at 4 &#176;C) and remove the supernatant. Add 200 &#181;L of CD11b-microbead solution (step 1.5) per tube and resuspend carefully (Figure 2I).</li>
	<li>Incubate the tubes for 15-20 min at 4 &#176;C. Carefully resuspend the pellet with 6 mL of sorting buffer (Figure 2I-J).</li>
	<li>Centrifuge at 300 x g for 10 min&#160;(at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 8 mL of sorting buffer (Figure 2K).</li>
	<li>Follow the POSSEL program on the separator (see Table of Materials) to prepare eight columns. Transfer cells 1 mL by 1 mL on the column. Wait for all the cells to pass through before adding another mL. Elute CD11b+ cells on sterile elution plate with 1 mL of sorting buffer (Figure 2L).</li>
	<li>Pool CD11b+ cells in a new 50 mL tube (Figure 2M).</li>
	<li>Centrifuge at 300 x g for 10 min&#160;(at 4 &#176;C) and remove the supernatant. Carefully resuspend the pellet with 10 mL of cold microglia medium (step 1.6) (Figure 2N).</li>
	<li>Count the CD11b+ cells. At P8, one should obtain ~650,000 cells per brain. 
	NOTE: In the present protocol, the cells were counted using an automated cell counter (see Table of Materials).</li>
	<li>Resuspend the cells in cold microglia medium to a final concentration of 650,000-700,000 cells/mL and dispense in cell culture plates. 
	NOTE: The 6-well plates are for Western Blot&#160;(2 mL per well); the 12-well plates are for RT-qPCR analysis (1 mL per well), and the 96-well plates are for phagocytic assay (250 &#181;L per well); all three were used for this work. However, in this manuscript, the results from the Western Blot analysis are not shown, but it is possible to perform with this protocol.</li>
	<li>Place the plates at 37 &#176;C with 5% CO2&#160;overnight. Change the medium carefully with a 1,000 &#181;L pipette with pre-heated microglia medium.</li>
	<li>Place the plates at 37 &#176;C with 5% CO2&#160;overnight before stimulation. 
	NOTE: Isolating microglia from more than 36 pups and after P9 are not recommended. This will increase the risk of contamination and accumulation of cell debris to activate microglia.</li>
</ol>
4.&#160;Cell stimulations
<ol>
	<li>Prepare stimulation reagents according to Table 3 using the commercially available reagents (see Table of Materials).</li>
	<li>Add the appropriate volume in each stimulated well. 
	NOTE: The appropriate volume depends on the concentration of the stimulus and the size of the well.</li>
	<li>Place the plates at 37 &#176;C with 5% CO2until the end of stimulation at 6 h, 24 h, or 48 h.</li>
	<li>For the Western Blot analysis, aspirate the supernatant and add 50 &#181;L of protein lysis buffer (see Table of Materials) with a pipette. Using tips, scratch the bottom of the plate to detach the lysed cells and transfer them to 1.5 mL tubes. Store at -80 &#176;C. 
	NOTE: The culture plates can be stored for years at -80 &#176;C if the supernatant is aspirated correctly.</li>
	<li>For RT-qPCR analysis6,31, aspirate the supernatant and store the culture plates directly at -80 &#176;C until mRNA extraction (step 5).</li>
	<li>For the phagocytic assay, please see step 6.</li>
</ol>
5. mRNA extraction and RT-qPCR analysis
<ol>
	<li>Perform RNA extraction, RT-PCR, and RT-qPCR following the manufacturer&#39;s protocol (see Table of Materials). Refer to Table 4 for the primer sequences5,6,31.</li>
	<li>Perform RT-qPCR analysis by following the previously published report5.</li>
</ol>
6. Phagocytic assay
<ol>
	<li>Stimulate the cells and perform phagocytic assay during the last 3 h of the stimulation. For example, for stimulation of 6 h, start the phagocytic assay after 3 h of stimulation; and for stimulation of 12 h, start the phagocytic assay 9 h after the beginning of the stimulation.</li>
	<li>Calculate the number of beads needed to prepare considering the ratio (1 cell: 50 beads). For one well of the 96-well plate, 8.1 x 106 beads are required. Prepare the beads mixture according to Table 5. 
	NOTE: Carefully vortex the beads stock vial before adding to the PBS/FBS mixture.</li>
	<li>Incubate for 1 h in a water bath at 37 &#176;C. Vortex every 10 min.</li>
	<li>Calculate the solution volume to add to each well. Add the solution and incubate for 3 h.</li>
	<li>Rinse the wells three times with 1x PBS. Read the fluorescence emission at 550 nm (Cy3 emission wavelength).</li>
</ol>
7. Purity quality control
<ol>
	<li>Evaluate the purity of CD11b magnetic isolation by flow cytometry (FACS) (before and after cell sorting), and then perform the RT-qPCR.
	<ol>
		<li>Count the cells and resuspend in FACS buffer (PBS + 2 mM of EDTA + 0.5% of Bovine Serum Albumin) in order to obtain a dilution of 10 x 106 cells/mL after step 3.5 and step 3.14.</li>
		<li>After 15 min of the conventional Fc blocking32,33, incubate the cells for 15 min with viability probe (FVS780) and fluorophore-conjugated antibodies against mouse CD45, CD11b, CX3CR1, ACSA-2, O4 or their corresponding control isotypes32,33&#160;at the concentration recommended by the manufacturers (see Table of Materials).</li>
		<li>Wash the cells with FACS buffer, fix, and permeabilize with a commercially available permeabilization kit (see Table of Materials).</li>
		<li>Perform Fc blocking again on the permeabilized cells, and then incubate with fluorophore-conjugated antibody against NeuN (see Table of Materials) or its control isotype for 15 min.</li>
		<li>Perform FACS analysis after washing with FACS buffer.</li>
		<li>After excluding the doublets and the dead cells based on morphological parameters and FVS780 staining, respectively, select the gating strategy for the surface expression of CX3CR1 (microglia), ACSA-2 (astrocytes), O4 (oligodendrocytes), and intracellular presence of NeuN (neurons) for the analysis of the percentage of positive cells.</li>
	</ol>
	</li>
	<li>Following step 5, extract mRNA and perform RT-qPCR to quantify Itgam (CD11b), Cx3cr1, Olig2, Synaptophysin, and Gfap mRNA. Normalize Cq using Rpl13a mRNA as a reporter, and perform a relative expression to Itgam mRNA.</li>
</ol>

The authors declare no conflicts of interest.

The current work presents a primary microglial cell culture using magnetically sorted CD11b+ cells. In addition to the microglial functional evaluation (RT-qPCR and phagocytic assays), microglial culture purity was also determined.
Classical microglia cell cultures are commonly generated from P1 or P2 rodent neonate brain and co-culture with astrocytes for at least 10 days. Microglia are then separated mechanically using an orbital shaker. The method to isolate and culture microglia in vit...

Microglia are the central nervous system resident macrophage-like cells derived from erythropoietic precursors of the yolk sac that migrate to the neuroepithelium during early embryonic development1. Apart from their immunity functions, they also play a significant role during neurodevelopment, particularly for synaptogenesis, neuronal homeostasis, and myelination2. In adulthood, microglia develop long cellular processes to scan the environment continuously. In case of homeostasis ruptures such as brain injury or brain disease, microglia can change their morphological appearance to adopt an amoeboid shape, migrate to the injured area, increase and release many cytoprotective or cytotoxic factors. Microglia have heterogeneous activation states depending on their developmental stage and the type of injury sustained3,4,5. In this study, these activation states are broadly classified into three different phenotypes: pro-inflammatory/phagocytic, anti-inflammatory, and immuno-regulatory, keeping in mind that in reality, the situation is likely to be more complex6.
Studying in vivo microglial activation and screening for neuroprotective strategies at early stages of brain development can be challenging due to (1) the fragility of animals before weaning and (2) the low number of microglial cells. Therefore in vitro studies on microglia are widely used for toxicity7,8,9, neuroprotective strategies5,10,11,12,13,14, and co-cultures15,16,17,18,19,20,21. In vitro studies can use either microglial cell lines, stem-cell-derived microglia, or primary microglia culture. All these approaches have advantages and disadvantages, and the choice depends on the initial biological question. The benefits of using primary microglia cultures are the homogenous genetic background, pathogen-free history, and control of the time when the microglia are stimulated after animal death22.
Over the years, different methods (flow cytometry, shaking, or magnetic labeling) were developed for culturing primary microglia from rodents, both neonate and adult23,24,25,26,27,28,29. In the present work, microglia isolation from mouse neonate pups is performed using previously described magnetic-activated cell sorting technology using microbead-coated anti-mouse CD11b25,27,29. CD11b is an integrin-receptor expressed at the surface of myeloid cells, including microglia. When there is no inflammatory challenge within the brain, almost all CD11b+ cells are microglia30. Compared to other previously published methods23,24,25,26,27,28,29, the present protocol balances immediate ex vivo microglial activation analyses and common in vitro primary microglial culture. Thus, microglia are (1) isolated at postnatal day (P)8 without myelin removal, (2) cultured without serum, and (3) exposed either to siRNA, miRNA, pharmacological compound, and/or inflammatory stimuli only 48 h after brain isolation. Each of these three aspects makes the current protocol relevant and rapid. First of all, the use of pediatric microglia allows obtaining dynamic and reactive viable cells in culture without requiring an additional demyelination step that could potentially modify microglial reactivity in vitro. The present protocol aims to get as close as possible to the physiological environment of microglia. Indeed, microglia never encounter serum, and this protocol does not require the use of serum either. Moreover, exposing microglia as early as 48 h after culture prevents them from losing their physiological faculties.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti mouse ACSA-2 PE Vio 615</td>
			<td>Miltenyi Biotec</td>
			<td>130-116-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse CD11b BV421</td>
			<td>Sony Biotechnology</td>
			<td>1106255</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse CD45 BV510</td>
			<td>Sony Biotechnology</td>
			<td>1115690</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse CX3CR1 PE Cy7</td>
			<td>Sony Biotechnology</td>
			<td>1345075</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti mouse NeuN PE</td>
			<td>Milli-Mark</td>
			<td>FCMAB317PE</td>
			<td></td>
		</tr>
		<tr>
			<td>anti mouse O4 Vio Bright B515</td>
			<td>Miltenyi Biotec</td>
			<td>130-120-016</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm permeabilization kit</td>
			<td>BD Biosciences</td>
			<td>554655</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b (Microglia) MicroBeads, h, m</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica TCS SP8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (10x)</td>
			<td>Thermo Scientific</td>
			<td>14200067</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E1644</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell culture 12-well plate, flat bottom + lid</td>
			<td>Dutscher</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell culture 96-well plate, flat bottom + lid</td>
			<td>Dutscher</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>Dutscher</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc blocking reagent (Mouse CD16/32)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon ECLIPSE TE300</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes (4 x 25 tubes)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10x</td>
			<td>Thermo Scientific</td>
			<td>14065049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS) -CaCl2 -MgCl2 10x</td>
			<td>Thermo Scientific</td>
			<td>14185045</td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR&#160;Green Supermix</td>
			<td>Bio-rad</td>
			<td>1725006CUST</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscript c-DNA synthesis</td>
			<td>Bio-rad</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex beads, amine-modified polystyrene, fluorescent red</td>
			<td>Sigma-Aldrich</td>
			<td>L2776-1mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS) from&#160;Escherichia coli&#160;O55:B5</td>
			<td>Sigma-Aldrich</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage-SFM serum-free medium</td>
			<td>Thermo Scientific</td>
			<td>12065074</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (70 &#956;m), 4 x 25 pcs</td>
			<td>Miltenyi Biotec</td>
			<td>130-110-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 PE</td>
			<td>Millipore</td>
			<td>MABC002H</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG2a PE Cy7</td>
			<td>Sony Biotechnology</td>
			<td>2601265</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL1 beta</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-684</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-24 Column Blocks</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-691</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiMACS Cell24 Separator</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit - Papain</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleocounter NC-200</td>
			<td>Chemometec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin RNA Plus XS</td>
			<td>Macherey Nagel</td>
			<td>740990.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nun EZFlip Top Conical Centrifuge Tubes</td>
			<td>Thermo Scientific</td>
			<td>362694</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTILUX Petri dish - 100 x 20 mm</td>
			<td>Dutscher</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>P&#233;nicilline-streptomycine (10 000 U/mL)</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2b, k BV421</td>
			<td>BD Biosciences</td>
			<td>562603</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2b, k BV510</td>
			<td>Sony Biotechnology</td>
			<td>2603230</td>
			<td></td>
		</tr>
		<tr>
			<td>REA control (S) PE vio 615</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-616</td>
			<td></td>
		</tr>
		<tr>
			<td>REA control (S) Vio Bright B515</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-445</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IFN-gamma Protein</td>
			<td>R&#38;D System</td>
			<td>485-MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-10 Protein</td>
			<td>R&#38;D System</td>
			<td>417-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-4 Protein</td>
			<td>R&#38;D System</td>
			<td>404-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Viability probe (FVS780)</td>
			<td>BD Biosciences</td>
			<td>565388</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic isolation of microglial cells from neonate mouse for primary cell cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Microglia is the CNS resident macrophage that gets activated when exposed to environmental challenges (trauma, toxic molecules, inflammation )4,5,6,34&#160;(Figure 3A). In vitro studies on microglia are commonly used to evaluate cell-autonomous mechanisms related to those environmental challenges and characterize activation state after pharmacological or g...

Watch this Scientific Journal Video about Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures at JoVE.com

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

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2.9K Views.  Université de Paris. Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Video: Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...