The following procedures have been approved by the Animal Ethics Committee at the Monash University. Healthy untreated neonate C57Bl6/J P3-6 mice were used to generate the representative results.
1. Enzymatic Digestion
NOTE: It is important to consider sterility when isolating and culturing primary cells. Whilst ensuring the environment is as sterile as possible, the initial dissection and harvest of murine brains can be completed outside of a laminal flow hood, with ...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+injury+in+premature+infants:+a+complex+amalgam+of+destructive+and+developmental+disturbances+(Report).">Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances (Report).</a> Lancet Neurology. 8 (1), 110 (2009).</li><li>Saliba, E., Henrot, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+Mediators+and+Neonatal+Brain+Damage.">Inflammatory Mediators and Neonatal Brain Damage.</a> Biology of the Neonate. 79 (3-4), 224-227 (2001).</li><li>Uwe-Karsten, H., Helmut, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+active+sensor+and+versatile+effector+cells+in+the+normal+and+pathologic+brain.">Microglia: active sensor and versatile effector cells in the normal and pathologic brain.</a> Nature Neuroscience. 10 (11), 1387 (2007).</li><li>Cherry, J. 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Microglia have the ability to be both pro- and anti-inflammatory, altered by environment stimuli. Previous studies have shown the modulation of microglia activation can confer neuroprotection. Their ability to provide protection to neurons and repair injury necessitates more research to further the current understanding of these complex cells. Thus, isolation of high purity primary microglia is an important and useful technique. This is a relatively quick method to obtaining highly pure primary microglia ready for in...

Damage acquired during the perinatal period from inflammation, hypoxic-ischaemia and haemorrhage can have an array of long term sequelae. The complex pathophysiology of perinatal brain injury is theorized to involve inflammation and ischemia with ensuing neuronal and axonal death1. The innate immune response plays an important role in the cascade of events leading to injury2.
Microglia, the resident immune cells within the central nervous system (CNS), are the first responders to injury3. Microglia are plastic cell types with the capacity to be both protective or toxic, dependent on the environment4. They are involved in chemotaxis, phagocytosis, antigen presentation and production of cytokines and reactive oxygen species4,5. Senescent microglia constantly survey the environment and are activated by the presence of a foreign or harmful substance4. Activation leads to a pro-inflammatory response, critical in CNS protection4. These M1 &#34;pro-inflammatory&#34; phenotype microglia are primarily involved in antigen presentation and death of pathogens4. Despite the crucial role of the inflammatory response in neuroprotection, uncontrolled or prolonged inflammation can be harmful and lead to neuronal damage4. However, when exposed to certain environmental stimuli, microglia can exhibit an anti-inflammatory phenotype. These pro-reparative M2 microglia have a critical role in wound healing and repair6, releasing a range of cytokines and other soluble mediators that downregulate inflammation, increase phagocytosis and promote repair4,7. The roles of microglia are diverse and include driving oligodendrocyte differentiation during re-myelination8, protecting neurons during oxygen and glucose depletion in stroke models9 and promoting neurite outgrowth in spinal cord injury models10.
The study of these glial cells represents an important aspect in understanding and manipulating the response to neuroinflammation. The described protocol allows for further investigation into the therapeutic potential of microglia modulation in neuroinflammatory disorders.
The modulation of microglial activation towards a neuroprotective role has been observed in a range of conditions11,12,13. Thus, improving current understanding and further studying modulation of microglial activation is critical, requiring the use of various models including both in vitro and in vivo. In vitro studies represent an important tool due to their greater efficiency, lower cost and ability to investigate an isolated cell population.
There are a range of protocols described in the literature for the isolation of microglia from murine brains, the challenge to efficiently produce a high yield sample with good viability and high purity. Commonly used methods of isolation of primary microglia are by magnetic separation and prolonged shaking of mixed glial cultures. Through personal experience, it was found that there was a high degree of cellular debris which obstructed the magnetic column. Thus, the following protocol was utilized, which incorporates an initial density gradient centrifugation step followed by CD11b magnetic separation. The protocol described below has been optimized to produce a highly pure sample in sufficient quantity. It is advantageous due to its high purity and the short time period &#8212; one can perform assays within 2 days without having to culture for 2-3 weeks. This protocol can potentially be adapted for the isolation of primary murine astrocytes.

<table border="0" cellpadding="0" cellspacing="0" width="929">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">DMEM, low glucose, pyruvate</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:344px;">11885084</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">DNaseI grade II from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Papain from papaya latex, buffered aqeuous solution</td>
			<td>Sigma-Aldrich</td>
			<td>P3125-100mg</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Hank&#39;s Balanced Salt Solution (1X)</td>
			<td>Gibco</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Hank&#39;s Balanced Salt Solution (10X)</td>
			<td>Gibco</td>
			<td>14185052</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep Mouse CD11b Positive Selection Kit</td>
			<td>StemCell Technologies</td>
			<td>18770</td>
			<td>EasySep magnet variant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep Buffer</td>
			<td>StemCell Technologies</td>
			<td>20144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Dulbecco&#39;s Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>14040182</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Trypsin (2.5%) (10X)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&#8482;)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Falcon 5mL Round Bottom High Clarity PP Test Tube, with Snap Cap, Sterile</td>
			<td>Corning</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">175cm&#178; Angled Neck Cell Culture Flask with Vent Cap</td>
			<td>Corning</td>
			<td>431080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Lipopolysaccharides from Escherichia coli O127:B8</td>
			<td>Sigma-Aldrich</td>
			<td>L5024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">96 Well TC-Treated Microplates size 96 wells, clear, polystyrene, round bottom</td>
			<td>Corning</td>
			<td>CLS3799</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Paraformaldehyde (powder, 95%)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Rabbit Anti-Iba1</td>
			<td>Wako</td>
			<td>01919741&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">FACS Antibodies</td>
			<td style="width:133px;">Company</td>
			<td style="width:344px;">Catalog Number</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">V450,Rat,Anti-Mouse,CD45,30-F11,RUO</td>
			<td style="width:133px;">BD Biosciences</td>
			<td>560501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">PerCP-Cy5.5 CD11b&#160;</td>
			<td>eBiosciences</td>
			<td>45-0112-82</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">ZombieNIR</td>
			<td>Biolegend</td>
			<td>423105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">pHrodo Red E. coli BioParticles Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>P35361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Annexin.V_FITC</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Propodium Iodide solution</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization and isolation of mouse primary microglia by density gradient centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

Using the methods outlined here, pure populations of microglia can be isolated and can be ready for characterization using in vitro and FACS analysis. To begin with, up to 18 animals can be used per cull, with an expected yield of approximately 450,000 - 600,000 microglial cells. It is crucial to first confirm the purity of the isolated cells, and to do so FACS analysis was performed by staining for the two markers CD45 and CD11b. Identification of microglia can prove troublesome...

Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

The overall goal of this protocol is to isolate a high-purity population of microglia from rodents. This method can help answer key questions in the neuroinformation field such identifying the immunomodulatory properties of stem cells on microglia or performing drug screening assays. The main advantage of this technique is the isolation of a high-purity population of microglia without the need for prolonged time in culture.To begin this procedure, insert the tips of the scissors through the opening in the spinal canal and make lateral incisions to the ear canal. Then, gently slide the tissue towards the rostrum to expose the skull. Next, make an incision along the sagittal suture towards the bregma.Then, insert the tip of the scissors into the bregma and make lateral incisions. Afterward, gently peel away the skull to expose the brain. Using curved forceps, remove it and transfer to a five-milliliter sterile container.Wash it two to three times with five milliliters of ice-cold wash media per wash to remove the blood. In a laminar airflow hood, place the contents of the five-milliliter container in a small Petri dish resting on ice. Using a sterile scalpel blade or sterile scissors, remove the cerebellum and olfactory bulb.Then, make a midline cut to separate the two hemispheres. Subsequently, identify the meningeal layer as a very thin layer of cells with a red tinge on the surface of the brain with visible blood vessels. Carefully peel away the meningeal layer with fine forceps taking care not to damage the cortices.If the meningeal layer breaks, continue peeling away the torn fragments until it is completely removed. Then, transfer the hemispheres to a new small Petri dish on ice and fill it with wash media. Chop the brain into small pieces with sterile scalpel blade.Following that, add 100 microliters of papain and 150 microliters of DNase1 into the media and incubate for 30 minutes at 37 degrees Celsius. After digestion, triturate the tissue using a P1000 pipette. If the tissue pieces are too large to enter the pipette, consider using a pair of sterile scissors to widen the pipette tip.During this process, take care not to introduce bubbles into the media as this may reduce cell viability. In a laminar flow hood, prepare a 50-milliliter conical tube with a 100-micrometer cell strainer. Pour the digest medium and brain pieces onto the strainer.Push through the pieces of brain by using the plunger of a sterile three-milliliter syringe in a grinding motion until there is no more tissue visible. Continuously wash the filter with the wash media throughout this process. This will wash through any cells trapped in the strainer.After that, centrifuge the single-cell suspension at 500 G for five minutes at four degrees Celsius. Then, prepare the stock isotonic Percoll by adding nine-to-one ratio of density gradient medium to 10X sterile HBSS. Prepare gradients as 30%SIP in DMEM and 70%SIP in one-X HBSS.For example, to prepare 10 milliliters of 30%SIP, add three milliliters of SIP to seven milliliters of DMEM. Next, aspirate the supernatant from the conical tube and re-suspend the cell pellet with eight milliliters of 30%SIP in DMEM. Transfer the full volume to a fresh 15-milliliter conical tube.Afterward, underlay the 70%SIP solution by filling a transfer pipette with 70%SIP and carefully push through the transfer pipette to the bottom of the conical tube. Once the tip is close to the bottom, gently push the contents through the transfer pipette. Subsequently, centrifuge the SIP layers including the cells at 650 G with brake zero and acceleration four for 25 minutes at room temperature.The 70%Percoll layer must be underlaid with great care. Disruption of this interface can severely impact the trapping of the microglia in the cell layer and severely reduce yields. Then, aspirate four milliliters of the media with cellular debris from the top of the tube to ease mononuclear cell removal in the following step.Next, carefully lower the pipette tip towards the interface and isolate the mononuclear cells from the 30/70 density gradient medium interface. Collect approximately three milliliters from the cloudy interface and transfer it to a new 15-milliliter conical tube. Following this, dilute the mixture with nine milliliters of HBSS to aid removal of the density gradient medium.Centrifuge the diluted density gradient medium interface containing the mononuclear cells at 500 G for five minutes. Aspirate the supernatant and re-suspend it with one milliliter of growth medium. Subsequently, stain the re-suspended cells with Trypan blue and perform a cell count using a hemocytometer.In this step, centrifuge the collected mononuclear cells at 300 G for 10 minutes at four degrees Celsius to remove the growth medium as this will interfere with the magnetic isolation. Using the same cell count obtained earlier, re-suspend at one times 10 to the eight nucleated cells per milliliter in PBS containing 2%FBS and one millimolar EDTA within a volume range of 0.1 to 2.5 milliliters. Next, add the full volume of nucleated cells in PBS to a fresh five-milliliter polystyrene round-bottom tube.Then, add 50 microliters of CD11b PE labeling reagent per one milliliter of sample. Incubate it at room temperature for 15 minutes protected from light. After that, add 70 microliters of selection cocktail per one milliliter of sample.Incubate it at room temperature for 15 minutes protected from light. Subsequently, mix the magnetic particles by pipetting up and down more than five times. Add 50 microliters per milliliter of to the sample and incubate at room temperature for 10 minutes protected from light.If the total volume of cell mixture is less than 2.5 milliliters, top up to this volume with PBS containing 2%FBS and one millimolar EDTA and mix by gently pipetting two to three times. Then, place the tube into the magnet and incubate at room temperature for five minutes. In one continuous motion, fully invert the magnet containing the tube for two to three seconds to pour off the supernatant.Return the magnet to an upright position and remove the tube from the magnet. Following that, wash the remaining cells out from the column by repeating the procedures. Re-suspend the cells in a desired growth medium.Rinse the side of the collection vessel to collect cells from the sides of the tube and maximize the yields. As can be seen in this figure, the isolated primary microglia retain their spherical cell body and distinct ramified structure. Here are the representative facts plots of the isolated microglia.The combined staining of annexin V and propidium iodide allows the categorization of apoptotic, necrotic or live cells after stimulation with LPS. hAEC-conditioned medium significantly reduced microglia apoptosis relative to controls suggesting a form of protection on this cell type. Once mastered, this technique can be performed in six to eight hours if performed correctly.When performing this method, it's important to take great care in layering the Percoll as this step will have the greatest impact on yields. Following this technique, other procedures such as the phagocytic function assay, or co-culture with neurons, can be used to answer additional questions about the immunomodulatory properties of your cell type of choice on primary microglia. After its development, this technique paved the way for researchers in the field to discover how primary microglia can be modulated in perinatal brain injury.The implications of this technique extend toward the therapy and diagnosis of perinatal brain injury as it allows the characterization of a primary immune cell type known to be involved in neuroinformation that leads to motor and cognitive dysfunction. Though this method can provide insight into motor disorders such as cerebral palsy, it can also be applied into other disorders where microglia plays a role in the pathogenesis such as Alzheimer's disease. We first had the idea for this method when we wanted a technique for the quick characterization of microglia circumventing potential epigenetic changes after prolonged time in culture.After watching this video, you should have a good understanding of how to isolate high-purity population of microglia for downstream characterization.

characterization-isolation-mouse-primary-microglia-density-gradient

Research

JoVE Journal

Neuroscience

19.0K vistas.  Hudson Institute of Medical Research. The overall goal of this protocol is to isolate a high-purity population of microglia from rodents. This method can help answer key questions in the neuroinformation field such identifying the immunomodulatory properties of stem cells on microglia or performing drug screening assays. The main advantage of this technique is the isolation of a high-purity population of microglia without the need for prolonged time in culture.To begin this procedure, insert the tips of the scissors throug...