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 cel...

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.

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

This protocol allow us to culture microglia in conditions that closely mimics the in vivo characteristics. Using magnetic cell sorting technology, our protocol allow us to stimulate microglia only two day in vitro, without using any serum in the culture medium. To begin, use small scissors to cut the skin from the neck to the nose, following the sagittal suture of 15 to 20 millimeters.Insert the tips with the foramen magnum parallel to the skull. Cut from each side to the eyes. Cut between the eyes with small scissors to detach the skull and brain from the head.With two forceps, grab the skull close to the olfactory bulbs, and carefully tear the skull. With a razor blade, remove the cerebellum and olfactive bulb, and cut the brain into two pieces. Place the brain pieces in a Petri dish containing 40 milliliters of HBSS without calcium and magnesium.Prepare dissociation mixture according to this table. Transfer 12 brain pieces into a C tube for a total weight of 1.2 grams per dissociation tube. Then place C tubes on the dissociater with heating.Start the optimized NTDK program in the dissociater. Centrifuge for 20 seconds. Complete the mechanical dissociation by pipetting three times.Transfer the cells to four 15-milliliter tubes with attached strainers. Rinse the strainers with 10 milliliters of HBSS with calcium and magnesium. Centrifuge for 10 minutes, and remove the supernatant with a 10-milliliter pipette.Carefully add 10 milliliters of HBSS with calcium and magnesium, and re-suspend the pellet. Again, centrifuge and remove the supernatant. Re-suspend the pellet with six milliliters of sorting buffer.Repeat the centrifugation and discard the supernatant. Then add 200 microliters of CD11b microbead solution and incubate the tubes for 15 to 20 minutes at four degrees Celsius. After incubation, re-suspend the pellet with six milliliters of sorting buffer.Repeat the centrifugation and re-suspend the pellet with eight milliliters of sorting buffer. Next, follow the Possel program on the separator to prepare eight columns. Pass the cells through the column by adding one milliliter of cell suspension at a time.With one milliliter of sorting buffer, elute CD11b-positive cells on a sterile elution plate. Pool the cells in a 50-milliliter tube. Centrifuge and re-suspend the pellet with 10 milliliters of cold microglia medium.Count the CD11b-positive cells. Re-suspend the cells in cold microglia medium to get a final concentration of 650, 000 to 700, 000 cells per milliliter. Dispense the suspension in cell culture plates.Incubate the plates overnight at 37 degrees Celsius with 5%carbon dioxide. On the next day, replace the medium with preheated microglia medium, and repeat the overnight incubation. Stimulate the cells before this step, and perform phagocytic assay during the last three hours of the stimulation.Calculate the number of beads according to this table at a ratio of 50 beads per cell. Prepare the beads mixture. Incubate the tube for one hour in a water bath at 37 degrees Celsius.Vortex every 10 minutes. To each well, add the calculated volume of the bead solution and incubate for three hours. Using this approach, the phagocytic activity of microglia was evaluated after stimulation by pro-inflammatory factors, such as interleukin-1 beta, plus interferon gamma, or lipopolysaccharides.After stimulation for six to 24 hours, fluorescent Cy3 microglia beads were analyzed by confocal microscope. After six hours, microglia start to phagocyte Cy3 beads only under interleukin-1 beta plus interferon gamma condition. After 24 hours, there was an increase of Cy3 fluorescence for both kinds of stimulation, highlighting increased phagocytic activity.The purity of microglial culture was elevated by flow cytometry, which showed an increase in cell viability after sorting. Using flow cytometry and RT-qPCR cell population markers were analyzed before and after sorting CD11b-positive cells from mice brains at postnatal day eight. These analyses distinguished various brain cell populations, such as CX3CR135 for microglia, O4, or OLIG2 for oligodendrocytes, NeuN, or synaptophysin, for neurons, and ACSA-2, or GFAP for astrocytes.After cell sorting using CD11b antibody, only microglia were obtained. It is important to pipette very gently while adding the suspension to the column to prevent clogging and avoid mechanical cell death. After this method, transcriptomic proteomics, such as western blood, or Luminex, and even immunochemistry can be performed in order to see the effect of microglial stimulation.

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2.7K vues.  Université de Paris. Ce protocole nous permet de cultiver la microglie dans des conditions qui imitent étroitement les caractéristiques in vivo. En utilisant la technologie de tri cellulaire magnétique, notre protocole nous permet de stimuler la microglie seulement deux jours in vitro, sans utiliser de sérum dans le milieu de culture. Pour commencer, utilisez de petits ciseaux pour couper la peau du cou au nez, en suivant la suture sagittale de 15 à 20 millimètres.Insérez les pointes avec le foramen...