Name: characterization and isolation of mouse primary microglia by density gradient centrifugation
Uploaded: 2018-02-16T13:30:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

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 all subsequent steps performed within a laminar flow hood.
<ol>
	<li>Using sterile instruments, euthanize the mouse by cervical dislocation, decapitate the animal and rinse with 100% ethanol. Using small sterile scissors, make small incisions along the right and left sides of the head. At this age, the skin peels away easily. Using the tips of the curved forceps, gently slide the tissue towards the rostrum to expose the skull, including the Bregma.</li>
	<li>Insert the tip of scissors in the opening of the spinal canal and make a lateral incision to the ear canal. Make an incision along the sagittal suture to the Bregma, gently pointing the tip of the scissors upwards to prevent damaging the brain. Insert tip of scissors at Bregma and make lateral incisions to the right and left sides of the head along the coronal suture.</li>
	<li>Using forceps, gently peel away the skull to expose the brain. To do so, grasp the edges of skull exposed by the lateral incision, and gently pull the skull to the side. The skull should peel away easily. Using curved forceps, gently scoop under the brain to remove it.</li>
	<li>Place the brain in a 5 mL sterile container, wash 2-3 times with ice-cold wash media (low glucose Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin), approximately 5 mL media per wash, to remove blood.</li>
	<li>In a laminar airflow hood, place the contents of the 5 mL container in a small petri dish (35 mm), resting on ice. Using a sterile scalpel blade or sterile scissors, remove the cerebellum and olfactory bulb. Make a midline cut to separate the two hemispheres.</li>
	<li>Carefully peel away the meningeal layer with fine forceps, taking care not to damage the cortices. 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. Keeping the brain cold is essential for proper meninges removal. If the meningeal layer breaks, continue peeling away the torn fragments until it is completely removed.</li>
	<li>Transfer the hemispheres to a new small petri dish (on ice) and fill with wash media. Chop the brain into small pieces with sterile scalpel blade/scissors. 
	NOTE: These should be ~1 mm2 in size, and care should be taken not to cut them into too small pieces as this may reduce yield.</li>
	<li>Add 100 &#181;L papain (17 U/mg stock) and 150 &#181;L DNase I into the media and incubate for 30 min at 37 &#176;C</li>
	<li>Following digestion, triturate tissue using a P1000 pipette. If tissue pieces are too large to enter the pipette, consider using a pair or 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.</li>
</ol>
2. Myelin Debris Removal
<ol>
	<li>Under a laminar flow hood use sterile equipment, prepare a 50-mL conical tube with a 100 &#181;m cell strainer, for each brain. Pour contents of the petri dish, containing the digest medium and brain pieces onto a strainer. Push through the pieces of brain by using the plunger of a sterile 3 mL syringe in a grinding motion until there is no more tissue visible. Following digestion, the brain tissue should fall apart easily.</li>
	<li>Continuously wash the filter with the wash media, by topping up the cell strainer throughout this process. This will wash through any cells trapped in the strainer. 
	NOTE: This should be done until there is approximately ~15 mL in the tube. This is to ensure that the strainer is sufficiently washed through and to maximize the amount of cells collected.</li>
	<li>Centrifuge the single cell suspension at 500 x g for 5 min at 4 &#176;C. During this time, prepare the density gradient medium as described.
	<ol>
		<li>Prepare the stock isotonic percoll (SIP), which is the density gradient medium used. Do this by adding 9:1 ratio of density gradient medium to 10x sterile Hanks balanced salt solution (HBSS).</li>
		<li>Prepare gradients as 30% SIP in DMEM and 70% SIP in 1x HBSS. For example, to prepare 10 mL of 30% SIP, add 3 mL of SIP to 7 mL DMEM.</li>
	</ol>
	</li>
	<li>Aspirate the supernatant from the conical tubes and resuspend the cell pellet with 8 mL of 30% SIP in DMEM. Transfer the full volume to a fresh 15 mL conical tube.</li>
	<li>Underlay the 70 % SIP solution. To do so, fill 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 through the contents of the transfer pipette. 
	NOTE: Do this slowly so as not to disrupt the 30/70 interface. There must be a clear line separating the two layers.</li>
	<li>Centrifuge the SIP layers, including the cells, at 650 x g with brake 0 and acceleration 4, for 25 min at room temperature. 
	NOTE: It is essential to complete the spin with brake OFF and to allow the centrifuge to slowly come to a halt, as application of brake will disrupt the interphase and dramatically reduce the cell collection between the SIP layers.</li>
	<li>Aspirate the cellular debris at the top of the tube, and remove approximately 4 mL media from the top. This will ease the removal of the mononuclear cells in the following step. Using a P1000 pipette, carefully lower the pipette tip towards the interphase. Isolate the mononuclear cells from the 30/70 density gradient medium interface. Collect approximately 3 mL from the cloudy interface and transfer to a new 15 mL conical tube. Following this, dilute the mixture with 9 mL of HBSS to aid removal of the density gradient medium.</li>
	<li>Centrifuge the diluted density gradient medium interphase, containing the mononuclear cells at 500 x g for 5 min. Aspirate supernatant and resuspend with 1 mL growth medium (DMEM supplemented with 10 % fetal bovine serum and 1% antibiotics). Following this, stain the resuspended cells with trypan blue and perform a cell count using a haemocytometer.</li>
</ol>
3. Magnetic Activated Cell Sorting
NOTE: These steps are modified from manufacturers&#39; protocol.
<ol>
	<li>Centrifuge the collected mononuclear cells at 300 x g for 10 min at 4 &#176;C to remove the growth medium as this will interfere with the magnetic isolation. Using the same cell count obtained from Step 2.8, resuspend at 1 x 108 nucleated cells/mL in PBS (Ca++ and Mg++ free) containing 2 % FBS and 1 mM EDTA, within a volume range of 0.1-2.5 mL.</li>
	<li>Add the full volume of nucleated cells in PBS from the previous step to a fresh 5 mL (12 x 75 mm) polystyrene round bottom tube. Add 50 &#181;L CD11b PE labelling reagent per 1 mL of sample. Incubate at room temperature for 15 min protected from light.</li>
	<li>Add 70 &#181;L of selection cocktail (a combination of monoclonal antibodies against CD11b in PBS) per 1 mL of sample. Incubate at room temperature for 15 min protected from light.</li>
	<li>Mix magnetic particles by pipetting up and down more than 5 times. Add 50 &#181;L/mL to the sample. Incubate at room temperature for 10 min protected from light. 
	NOTE: These particles then form a tetrameric complex with the antibodies and will get attached to the magnetic column.</li>
	<li>If the total volume of cell mixture is less than 2.5 mL, top up to this volume with PBS (Ca++ and Mg++ free) containing 2 % FBS and 1 mM EDTA, and mix by gently pipetting 2-3 times. Place the tube (without lid) into the magnet and incubate at room temperature for 5 min.</li>
	<li>In one continuous motion, fully invert the magnet containing the tube for 2-3 s, pouring off the supernatant. Return the magnet to an upright position, and remove the tube from the magnet. Prepare to wash the remaining cells out from the column. 
	NOTE: The supernatant contains unlabelled and unwanted cells, which are removed by inverting the tube while still in the magnet. The tube contains the attached Cd11b+ cells.</li>
	<li>Wash the cells by repeating steps 3.5 and 3.6 twice more. If the sample is greater than 1 mL, a further repeat of steps 3.5 and 3.6 is recommended.</li>
	<li>Resuspend the cells in a desired growth medium. Rinse the side of the collection vessel as well (e.g. a 15 mL tube) to collect cells from the sides of the tube and maximize yields.</li>
</ol>
4. Verification of Microglia Purity
NOTE: The primary microglia isolated from 3 L (n = 5 animals total) were verified via fluorescent activated cell sorting (FACS) to determine purity for the representative results.
<ol>
	<li>Culture cells in a T-25 flask in the growth medium containing 10% FBS overnight, seeding at a density of 1 - 2 x 106 cells per flask.</li>
	<li>Wash the cells in the T-25 flasks with PBS 3x for 5 min to remove any remaining growth media which might interfere with the trypsinization.</li>
	<li>Add 2 mL of 0.25% trypsin to detach the cells from the flasks. Ensure that the volume of trypsin is sufficient to cover the entire surface of the flask. Following gentle swirling of the flask to ensure uniform coverage of trypsin, incubate for 5 min at 37 &#176;C.</li>
	<li>Quench the trypsinization process by adding 2 mL of growth media containing 10% FBS into the flask.</li>
	<li>Collect the supernatant containing the trypsinized cells and centrifuge at 500 x g for 5 min at 4 &#176;C to collect cells.</li>
	<li>Remove the supernatant containing the growth medium and trypsin and resuspend the pellet in 500 &#956;L FACS buffer. Perform the cell count using trypan blue.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Perform Fc receptor block by adding 50 &#956;L FACS buffer + 1 &#956;L FcR blocker per 5 x 106 cells. Incubate for 15 min at 4 &#176;C. 
	NOTE: The Fc receptor block is a critical step in the staining procedure to prevent non-specific binding of immunoglobulins to Fc receptors in immune cell populations. This blockade does not affect downstream binding of other antibodies.</li>
	<li>Terminate the blocking process by diluting with 500 &#956;L FACS buffer. Following this, centrifuge the mixture containing the cells at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend in fresh 500 &#956;L FACS buffer.</li>
	<li>Place the majority of cells (e.g. if resuspending the cells in 500 &#956;L of FACS buffer, use 400 &#956;L) into a sample tube. Distribute the remaining cells amongst the control tubes and top up to 100 &#956;L per control tube (e.g. for 6 control tubes, put 16.67 &#956;L into each control tube and top up with 83.33 &#956;L FACS buffer). Make the groups (control tubes in bold) as Unstained, single stained (CD45, CD11b), single live/dead stain, FMOs, and sample tubes.</li>
	<li>Pellet the cells in all tubes by centrifuging at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Stain the cells in the tube with 1 &#956;L antibody per 100 &#956;L FACS buffer per 5 x 106 cells. Incubate for 20 min at 4 &#176;C. 
	NOTE: Use 50 &#956;L antibody cocktail if less than 2 x 106 cells are used.</li>
	<li>Wash the cells with 500 &#956;L FACS buffer. Centrifuge the cells at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cells in 300 &#956;L FACS buffer. 
	NOTE: Use ~100 &#956;l if cell count is less than 2 x 106.</li>
	<li>Using flow cytometry analysis, quantify the cells that are CD45low and positive for CD11b staining. This percentage corresponds to the isolated primary microglia.</li>
</ol>
5. Immunohistochemical Staining of Primary Microglia
<ol>
	<li>Plate the cells in a 96-well plate at a density of 1 x 105 cells with the growth medium and incubate overnight.</li>
	<li>Remove the supernatant and wash 3 times with PBS.</li>
	<li>Fix the cells with 4% paraformaldehyde in PBS for 15 min. Remove the PFA with a P1000 pipette, then wash them 3 times with PBS + 0.1 % Triton-X.</li>
	<li>Block for non-specific binding with 3% bovine serum albumin for 1 h at room temperature.</li>
	<li>Incubate with rabbit Iba-1 (1:100) for 24 h at 4 &#176;C. Following this, remove the primary antibody mixture and wash 3 times with PBS.</li>
	<li>Incubate with secondary anti-rabbit GFP conjugate (1:200) for 1 h at room temperature. Following this, remove the secondary antibody mixture and wash 3 times with PBS.</li>
	<li>Mount slides with a fluorescent mounting medium and capture images using a fluorescent microscope.</li>
</ol>
6. Quantification of Microglia Using the pHrodo Assay
NOTE: The pHrodo assay allows for identification of levels of phagocytosis in cultured cells. Upon uptake via endocytosis, internalization into the more acidic environment increases levels of fluorescence of the bioparticle conjugates. Fluorescence levels can then be quantified by FACS. The following steps are modified from manufacturers&#39; protocol.
<ol>
	<li>Culture the cells in a T-25 flask in the growth medium overnight, seeding at a density of 1 - 2 x 106 cells per flask. Wash the cells in T-25 flasks with PBS 3 times for 5 min.</li>
	<li>Detach the cells using 2 mL 0.25% trypsin. Incubate for 5 min at 37 &#176;C.</li>
	<li>Quench the trypsinisation process by adding 2 mL of growth media containing 10% FBS into the flask.</li>
	<li>Centrifuge the cell suspension at 500 x g for 5 min at 4 &#176;C to pellet cells.</li>
	<li>Remove the supernatant and resuspend the pellet containing the primary microglia in culture medium at 106 cells/mL. 
	NOTE: Alternatively, plate cells in a 96-well plate at least a day before and aim for 1 x 105 viable cells per well on the day the assay is to be performed.</li>
	<li>Plate cells into 96-well plate at 1 x 105 cells/well, 100 &#956;L per well. Plate controls and experimental wells in triplicate, and leave one empty well per positive control for a no-cell control background subtraction.</li>
	<li>Add 100 &#956;L of growth media to the wells left empty for no-cell background subtraction. 
	NOTE: Like any other in vitro assay, appropriate controls are vital for the accuracy of the readouts. Alongside the no-cell control (background reading), also include the negative control well (pHrodo conjugate added, but placed on ice).</li>
	<li>Cover the plate and incubate for 1 h in an incubator with 5 % CO2 at 37 &#176;C.</li>
	<li>Prepare experimental wells by adding stimulant and treatment. Add vehicle controls (growth media only) to untreated wells. 
	NOTE: Lipopolysaccharide 1 &#181;g/mL [stimulant] and human amnion epithelial cell (hAEC)-conditioned media [treatment] were used to generate the representative results.</li>
	<li>Thaw a vial of conjugated dye, add 2 mL uptake buffer and briefly vortex. Transfer the tube contents into a clean glass tube and sonicate for 5 min, to ensure that particles are evenly dispersed.</li>
	<li>Aspirate the culture medium from microplate wells and quickly replace with 100 &#956;L of dye suspension. Cover and incubate at 37 &#176;C for 1 h. 
	NOTE: Remember to stain the control tubes as well.</li>
	<li>Using flow cytometry analysis, quantify cells staining positive for the conjugated pHrodo beads and present as a percentage (of parent) of actively phagocytosing microglia.</li>
</ol>
7. Quantification of Microglia Apoptosis Following Inflammatory Insult
<ol>
	<li>Repeat the steps 1-7 from &#34;Verification of microglia purity&#34; section.</li>
	<li>Place the majority of cells (e.g. if you resuspended the cells in 500 &#956;L of FACS buffer, use 400 &#956;L) into a sample tube. Distribute the remaining cells amongst the control tubes and top up to 100 &#956;L per control tube (e.g. for 6 control tubes, put 16.67 &#956;L into each control tube and top up with 83.33 &#956;L FACS buffer). Groups (control tubes in bold): Unstained, single stains (AnnexinV, Propidium iodide), single live/dead stain, FMOs, sample tubes.</li>
	<li>Pellet the cells in all the tubes by centrifuging at 500 x g for 5 min.</li>
	<li>Incubate with 1 &#956;L antibody/100 &#956;L FACS buffer/5 x 106 cells for 20 min at 4 &#176;C. 
	NOTE: Use 50 &#956;L if less than 2 x 106 cells.</li>
	<li>Wash the cells by adding 500 &#956;L FACS buffer and centrifuge at 500 x g for 5 min.</li>
	<li>Resuspend the cell pellet in 300 &#956;L FACS buffer. 
	NOTE: ~100 &#956;L if cell count is less than 2 x 106.</li>
	<li>Quantify cells within each quadrant (Q1: necrotic, Q2: late apoptotic, Q3: viable, Q4: early apoptotic).</li>
</ol>

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The authors have nothing to disclose.

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 vitro experimentation within 2 days.
There are a range of protocols described in the literature for the isolation of primary microglia from murine brains. The principal challenge is to efficiently produce sufficient sample, high in viability and purity. The main advantage of this protocol is the yield of a highly pure sample without the need for the time in culture. Thus, the method is shortened from 2 weeks to 2 days, this facilitates fast results. The characterization steps yield robust functional data about microglia, enhancing current understanding of these complex cells. The protocol combines two current approaches - density gradient centrifugation and magnetic separation. Magnetic separation has been well validated in the literature for isolation of microglia in a relatively gentle manner compared to other isolation techniques14,15,16. Whilst there are undoubtedly changes to the functional characteristics of the isolated primary microglia compared to in their native state in vivo, any changes to microglia properties as observed in the assays developed above are calculated from a post-digestion baseline, and as such are reflective of direct modulation of this immune subset.
Whilst the protocol is relatively straightforward, care during critical steps will help to ensure a good yield. Firstly, it is vital to use low glucose media to support glial growth. Furthermore, the murine brains must be kept ice cold at all times during the isolation. Thus, it is recommended to pre-chill all medium, as well as instruments if possible, and perform the procedure on ice. Importantly, prolonged isolation times will lead to poorer cell health and yield, thus it is critical to work quickly. Moreover, proper removal of the meningeal layer is a crucial step when working with adult mice, otherwise the fibroblasts will outcompete the glial cells in terms of growth. During the enzymatic digestion of the brains, it is important not to cut the brains into too small pieces as this can increase cell death, ideally aim for 1 mm2 pieces of tissue. When grinding the tissue through the cell strainer, thoroughly rinse with media every few minutes to ensure all cells are washed through and do not get trapped in the strainer, additionally it is recommended to use a 100 &#956;m cell strainer, smaller filters result in a reduced yield. Another critical step is the underlaying of the 70% density gradient medium layer, performing this step carefully and slowly so as to ensure a clear interphase is seen is essential, the use of a Pasteur pipette is recommended. Also, the use of 15 mL conical tubes is recommended as the separation of cells was less effective when 50 mL conical tubes were utilized. Lastly, the density gradient centrifugation must be performed at room temperature, as temperature can affect the density gradient.
This protocol describes a reliable method of producing highly pure primary microglia cells as shown by both high expression of CD45 and positive expression of Cd11b (as compared to low CD45 expression seen in peripheral monocytes). Cells can also be isolated relatively quickly, thus it has the scope to significantly increased understanding of these glia cells. It can be used to identify different microglia populations from different backgrounds, allowing for studies which may reveal differences between microglia isolated from diseased/injured and healthy animals. Through isolation of a pure microglia population, therapeutic modulation of this immune cell population can be assessed. For instance, it was found that hAEC-conditioned media increased phagocytosis, as well as improved microglia survival during an inflammatory stimulus, thus affording therapeutic benefits17. This correlated to reduced apoptotic debris and thus suggests clearance of these debris may have neuroprotective effects.
The protocol can be applied to both neonatal or adult mice, however older mice will produce a reduced yield. Furthermore, it may be adapted to isolate primary astrocytes from neonatal or adult mice, again older mice will result in a decreased yield. Additionally, the modification of the mechanical dissociation step with a custom-made nylon mesh with larger pore sizes as opposed to the 100 &#956;m cell strainer, might further improve cell yield18.
Limitations of this protocol include the lower yield and the time required for this protocol. The yield is approximately 150,000 - 200,000 cells per cull of six animals, versus the millions that can be obtained through weeks of cell culture. Additionally, whilst cells can be obtained within two days, the procedure on the first day is quite time-consuming, taking approximately six hours.
Nonetheless, this method is a highly useful tool in understanding the role of microglia in disease, capable of isolating and characterising the cells within two days. It produces high purity primary microglia cells, facilitating accurate and efficient research.

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><tbody><tr><td>DMEM, low glucose, pyruvate</td><td>Gibco</td><td>11885084</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>DNaseI grade II from bovine pancreas</td><td>Sigma-Aldrich</td><td>10104159001</td><td></td></tr><tr><td>Papain from papaya latex, buffered aqeuous solution</td><td>Sigma-Aldrich</td><td>P3125-100mg</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Gibco</td><td>16140071</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (1X)</td><td>Gibco</td><td>14175-103</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (10X)</td><td>Gibco</td><td>14185052</td><td></td></tr><tr><td>EasySep Mouse CD11b Positive Selection Kit</td><td>StemCell Technologies</td><td>18770</td><td>EasySep magnet variant</td></tr><tr><td>EasySep magnet</td><td>StemCell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Buffer</td><td>StemCell Technologies</td><td>20144</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline</td><td>Gibco</td><td>14040182</td><td></td></tr><tr><td>Trypsin (2.5%) (10X)</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&amp;trade;)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Falcon 5mL Round Bottom High Clarity PP Test Tube, with Snap Cap, Sterile</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>175cm&amp;sup2; Angled Neck Cell Culture Flask with Vent Cap</td><td>Corning</td><td>431080</td><td></td></tr><tr><td>Lipopolysaccharides from Escherichia coli O127:B8</td><td>Sigma-Aldrich</td><td>L5024</td><td></td></tr><tr><td>96 Well TC-Treated Microplates size 96 wells, clear, polystyrene, round bottom</td><td>Corning</td><td>CLS3799</td><td></td></tr><tr><td>Paraformaldehyde (powder, 95%)</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Rabbit Anti-Iba1</td><td>Wako</td><td>01919741&amp;nbsp;</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 488)</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>&lt;strong&gt;FACS Antibodies&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>V450,Rat,Anti-Mouse,CD45,30-F11,RUO</td><td>BD Biosciences</td><td>560501</td><td></td></tr><tr><td>PerCP-Cy5.5 CD11b&amp;nbsp;</td><td>eBiosciences</td><td>45-0112-82</td><td></td></tr><tr><td>ZombieNIR</td><td>Biolegend</td><td>423105</td><td></td></tr><tr><td>pHrodo Red E. coli BioParticles Conjugate</td><td>Thermo Fisher Scientific</td><td>P35361</td><td></td></tr><tr><td>Annexin.V_FITC</td><td>Miltenyi Biotech</td><td>130-093-060</td><td></td></tr><tr><td>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, as many markers expressed by microglia are also expressed by macrophages, and the use of these two markers allows accurate and reliable quantification. Specifically, microglia have a low expression of CD45, compared to the high expression seen in CNS and peripheral macrophages, and are also positive for CD11b (Figure 1). From this particular isolation, a purity of 89.3 % was reported. After staining for CD11b, we note that our primary microglia share similar morphological features, with a range of morphologies from the distinctly unramified (large spherical cell body with little or no extended processes) to the more ramified (with smaller, more oval somata and typically up to secondary order processes) correlating to a range of activation states, as expected to see in vivo (Figure 2).
Following this, two characterization assays for microglia function and survival were run. Microglia are the major phagocytic cell in the CNS, and the pHrodo assay allows quantification of this particular functional property. A near 3-fold increase in phagocytic function after 24-h co-culture with hAEC conditioned medium (Figure 3) was noted, as measured by the quantification of fluorescent pHrodo particles. Finally, an AnnexinV-PI staining following co-culture was performed to identify survival of microglia following an inflammatory insult. A reduction in microglia apoptosis after treatment with conditioned medium (Figure 4) was observed. These findings suggest that hAEC conditioned medium protects microglia and enhances their phagocytic activity, which might have therapy in perinatal brain injury and neuroinflammatory disorders.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57065/57065fig1.jpg" /> 
Figure 1: Expression of CD45 and CD11b by microglia. Each dot represents a labelled cell and FACS analysis allows elucidation of separate cell populations. Microglia have a lower expression of the CD45 antigen compared to peripheral monocyte-derived macrophages and are positive for CD11b. Numbers below antibody label indicate the proportion of gated cells expressed as percentage of the parent population. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57065/57065fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57065/57065fig2.jpg" /> 
Figure 2: Morphology of isolated primary microglia. As can be seen in this figure, microglia retain their spherical cell body and distinct ramified structure. Scale bar = 20 &#181;m.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57065/57065fig3.jpg" /> 
Figure 3: Quantification of phagocytic function in isolated primary microglia. pHrodo-labelled particles only fluoresce when in acidic environments, such as in cellular endosomes following phagocytosis. Here, an increase in pHrodo particle uptake in microglia treated with hAEC conditioned medium is observed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57065/57065fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57065/57065fig4.jpg" /> 
Figure 4: Analysis of microglia survival after inflammatory insult. (A) Representative FACS plots of isolated microglia. The combined staining of AnnexinV and propidium iodide allows categorisation into either apoptotic, necrotic, or live cells after stimulation with LPS. (B) hAEC conditioned medium significantly reduced microglia apoptosis relative to controls, suggesting a form of protection on this cell type. * signifies p &#60;0.05, student&#39;s t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57065/57065fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Wash medium</td>
		</tr>
		<tr>
			<td>Low Glucose Dulbecco&#39;s Modified Eagle Medium</td>
		</tr>
		<tr>
			<td>1% v/v penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>Digest medium (per brain)</td>
		</tr>
		<tr>
			<td>150 &#181;L DNase I&#160;</td>
		</tr>
		<tr>
			<td>100 &#181;L Papain (17U/mg stock)</td>
		</tr>
		<tr>
			<td>3 ml wash medium&#160;</td>
		</tr>
		<tr>
			<td>Growth medium</td>
		</tr>
		<tr>
			<td>Low Glucose Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
		</tr>
		<tr>
			<td>10% foetal bovine serum&#160;</td>
		</tr>
		<tr>
			<td>1% v/v penicillin-streptomycin&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Table of solutions.

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 ...

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

Nanyang Technological University

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JoVE Journal

Neuroscience

19.2K Views.  Hudson Institute of Medical Research. Een protocol voor de isolatie van primaire microglia vanuit lymfkliertest brains wordt gepresenteerd. Deze techniek helpt bij het bevorderen van het huidige begrip van neurologische aandoeningen. Dichtheid kleurovergang centrifugeren en magnetische scheiding worden gecombineerd om voldoende opbrengst van een zeer zuivere monster. Voorts schetsen we de stappen voor de karakterisering van microglia.

Video: Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Immunology and Infection

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 Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

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 ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Isolation of Hypothalamic Microglia from Mice Using MACS for In Vitro Assays

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

Author Spotlight: Investigating the Impact of Nutrition on Mouse Brain Function and Metabolic Disorders 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Magnetic Isolation of Microglia from Mouse Pup Brains

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

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

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting

Source: Zhang, H. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63511">Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination.</a> J. Vis. Exp. (2022).
This video demonstrates a protocol for extracting microglial cells from demyelinating lesions in mouse brains using magnetic cell sorting. The protocol involves dissecting the lesioned brain tissue containing microglial cells, treating the tissue with specific enzymes to obtain a single-cell suspension, and utilizing microglial receptor-specific magnetic beads to isolate the microglial cells.

 All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Materials

	&#160;Prepare ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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