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 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, 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

Research

JoVE Journal

Neuroscience

19.1K Views.  Hudson Institute of Medical Research. 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. 

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

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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

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

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

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

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

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.

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. ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...