This work was supported by the Christopher and Dana Reeve Foundation International Research Consortium on Spinal Cord Injury, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the JPB Foundation, the Novartis Institute of Basic Research, generous contributions from Vincent and Stella Coates, and the Damon Runyon Cancer Research Foundation (DRG-2125-12).

All procedures involving rodents conformed to Stanford University guidelines, which comply with national and state laws and policies. All animal procedures were approved by Stanford University&#39;s Administrative Panel on Laboratory Animal Care.
NOTE: All solution and buffer compositions are provided in the Table of Materials.
1. Prepare a Petri Dish for CD11b Immunopanning (Day 0)
NOTE: Prepare 1 immunopanning dish for every 1 - 2 juvenile rat brains.
<ol>
	<li>Add 25 mL of 50 mM Tris pH 9.5 solution to a 15-cm Petri dish.</li>
	<li>Add goat anti-mouse IgG (H+L chains) to the dish for a final concentration of 6 &#181;g/mL. Swirl plate to evenly distribute.</li>
	<li>Incubate the dish for 1 - 3 h at 37 &#176;C.</li>
	<li>Rinse dishes three times with DPBS++ (phosphate-buffered saline (PBS) with Ca2+ and Mg2+), then replace with a solution of panning buffer containing 1 &#181;g/mL OX42 antibody. Leave the dishes overnight at room temperature on a flat surface.</li>
</ol>
2. Tissue Collection (Day 1)
NOTE: This protocol should take ~3 - 4 h.
<ol>
	<li>Before beginning, ensure all solutions are sterile and chilled on ice. Chill all instruments on ice and sterilize with ethanol prior to use.</li>
	<li>Following appropriate regulatory procedures, sacrifice a juvenile laboratory rat by carbon dioxide asphyxiation. 
	NOTE: Alternatively, younger animals may be sacrificed with a lethal dose of ketamine/xylazine (100 - 200 &#181;L of 24 mg/mL ketamine, 2.4 mg/mL xylazine). Ketamine/xylazine must be used if animals are less than 14 days of age. If using multiple animals, extract the tissue from one animal and place it into pre-chilled DPBS++ on ice before proceeding to subsequent animals.</li>
	<li>Pinch a hindpaw and ensure complete unresponsiveness from the animal before proceeding.</li>
	<li>Transcardially perfuse the animal with 10 - 30 mL of ice-cold perfusion buffer using a 27&#189;-G needle until buffer runs clear. 
	NOTE: Volume of Perfusion buffer will vary depending on the size/age of the animal.</li>
	<li>Immediately after perfusion remove the head of the animal. Coming in from the spinal cord cut the occipital condyle on each side with dissection scissors, be careful not to damage the brain. After cutting each side carefully, cut up one side along the parietal and frontal bone towards the nasal bone. With forceps carefully pull back the top of the skull, quickly remove the brain (or CNS structures of interest), and place into pre-chilled DPBS++ on ice.</li>
	<li>Repeat for all remaining animals. 
	NOTE: All proceeding steps should be performed in a laminar flow hood under proper sterile conditions.</li>
</ol>
3. Mechanical Dissociation (Day 1)
<ol>
	<li>After all brains have been collected, chop one brain into 1 mm3 chunks in a Petri dish on ice with a cold scalpel blade, and transfer to an ice-cold dounce homogenizer with 5 - 7 mL ice-cold douncing buffer. Dissociate one brain at a time.</li>
	<li>Dissociate the tissue using 10 - 20 gentle and incomplete strokes with a loose-fitting dounce homogenizer. Take care not to directly crush the tissue at the bottom of the homogenizer, but instead impel the tissue through the space between the sides of the piston and the homogenizer.</li>
	<li>Carefully remove the piston to prevent introduction of air bubbles. Allow poorly dissociated tissue chunks to settle to the bottom of the homogenizer, and transfer supernatant to a new chilled 50-mL conical tube.</li>
	<li>Replace the removed volume with fresh douncing buffer, and repeat steps 3.2 and 3.3 for a total of 3 - 4 rounds, or until all tissue has been dissociated. Repeat the procedure for each brain.</li>
</ol>
4. Myelin Removal (Day 1)
NOTE: Myelin removal is used for isolation of microglia from animals older than P12.
<ol>
	<li>Measure the volume of the cell suspension in the 50-mL conical tube using a 25 mL pipette, then add ice-cold douncing buffer to adjust the total volume to 33.5 mL.</li>
	<li>Add 10 mL of myelin separation buffer (MSB) to the cell suspension and mix thoroughly by inverting the tube several times. This will result in a 23% final concentration of MSB in a volume of 43.5 mL.</li>
	<li>Centrifuge cells for 15 min at 500 x g at 4 &#176;C with slow braking. 
	NOTE: The centrifuge should take approximately 1.5 - 2 min to decelerate. This will generate an upper layer of myelin and dead cell debris, a somewhat murky supernatant, and a smaller pellet that is enriched for live cells.</li>
	<li>Remove the top layer of myelin/debris and the supernatant with a pipette. Take care when removing the top layer to ensure as much of it is removed as possible.</li>
	<li>Resuspend the cell pellet in 12 mL of panning buffer. Gently triturate the cell suspension to break up any clumps of cells that might remain.</li>
</ol>
5. Immunopanning (Day 1)
<ol>
	<li>Pass the cell suspension through a 70-&#181;m cell strainer to remove large debris or cell clumps.</li>
	<li>Rinse the OX42-coated panning dish three times with DPBS++. Don&#39;t allow the plate to completely dry between washes.</li>
	<li>Pour off the last DPBS++ wash and apply the filtered cell suspension to the panning dish. Gently swirl the plate to distribute the cells, then incubate the plate on a flat surface at room temperature for 20 min to allow cells to adhere. Do not incubate longer than 20 min or cells will become very difficult to recover from the dish.</li>
	<li>Rinse the panning dish with DPBS++ 10 times to remove non-adherent cells. Microglia will be firmly attached to the plate, so swirl the plate with each rinse to ensure removal of other non-adherent cells.</li>
	<li>Pour off the last DPBS++ wash and replace with 15 mL DPBS++ and 200 &#956;L trypsin (1.25% stock solution).</li>
	<li>Incubate the dish for &#8804;10 min at 37 &#176;C with 10% CO2 to trypsinize. 
	NOTE: Do not continue longer than 10 min or microglia will become difficult to remove.</li>
	<li>After 10 min of trypsinization, microglia will still be stuck to plate. Pour off trypsin/DPBS++ and gently wash 2x with DPBS++ to remove trypsin, replace with 12 mL of ice-cold microglia growth medium (MGM).</li>
	<li>Place panning dish on ice for 2 min to help weaken cell/substrate interaction, and make sure the dish is flat to prevent areas of the panning dish from drying out.</li>
	<li>Pipette vigorously with a 10-mL pipette and pipet controller on high speed to recover cells from the panning dish. Draw a 16 x 16 grid with the stream of liquid from the pipette to try and remove all the cells.</li>
	<li>Check cells under a microscope at 20X magnification to make sure cells have detached from plate. Mark spots on top of the dish where cells are still stuck, and repeat pipetting in those areas.</li>
	<li>Collect cell suspension and aliquot 3 - 4 mL of supernatant per 15-mL conical tube. Spin for 15 min at 500 x g at 4 &#176;C with slow braking. 
	NOTE: Spinning microglia through a small volume allows for the maximum recovery of cells.</li>
	<li>Aspirate the supernatant, leaving 0.5 mL of MGM with the cell pellet.</li>
	<li>Resuspend each pellet in remaining MGM, and pool the cells from all the tubes.</li>
	<li>Count cells with hemocytometer.</li>
	<li>Plate cells as described in Steps 6 - 7 depending on the application.</li>
	<li>Culture cells at 37 &#176;C with 10% CO2. 
	NOTE: Cells can be culture for up to 3 - 4 weeks with regular media changes or one week with no media changes.</li>
</ol>
6. Spot Coating Tissue Culture Plates/Coverslips (Day 1)
<ol>
	<li>Plate 15 &#181;L of collagen IV coating directly in the center of a 24-well anionic/cationic coated tissue culture plate (see Table of Materials for details) and incubate for 15 min at 37 &#176;C with 10% CO2.</li>
	<li>After counting cells with hemocytometer dilute cells to 2.3 x 105/mL in MGM. Aspirate collagen IV spot and immediately plate 15 &#181;L of cell suspension to this spot; this will give 3.5 x 103 cells/spot. Incubate for 5 - 10 min at 37 &#176;C with 10% CO2 to allow cells to adhere, add 500 &#181;L of CO2-equilibrated TGF-&#946;2/IL-34/cholesterol containing growth medium (TIC) gently to the well.</li>
	<li>If plating on coverslips, first coat sterile glass coverslips with 10 &#181;g/mL ploy-D-lysine (PDL) in H2O for 1 h at RT, wash 3 times with H2O, and let dry in a hood under UV light. Once coverslips are dry, proceed with spot plating on the coverslips as described in Step 6.2.</li>
</ol>
7. Plating for RNA/Protein Isolation
<ol>
	<li>On day 1, coat the entire area of a 24-well anionic/cationic coated tissue culture plate with collagen IV coating and incubate for 10 min -&#160;1 h at 37 &#176;C with 10% CO2, then remove and immediately plate 3 - 5 x 104 cells/well in CO2 equilibrated TIC media.</li>
	<li>On day 2, perform a 50% media change for each well the day after preparation. Dead cells tend to cluster in the center of the well, so take media from there.</li>
	<li>Perform a 50% media change every 2 - 3 days to maintain the cultures. If media changes introduce an unwanted variable for the cultures, cells can survive for approximately 1 week without any media changes, as opposed to over 3 weeks with regular maintenance.</li>
</ol>

<ol>
	<li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Function+in+the+Central+Nervous+System+During+Health+and+Neurodegeneration.">Microglia Function in the Central Nervous System During Health and Neurodegeneration.</a> Annu Rev Immunol. 35, 441-468 (2017).</li><li>Wu, Y., Dissing-Olesen, L., MacVicar, B. A., Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Dynamic+Mediators+of+Synapse+Development+and+Plasticity.">Microglia: Dynamic Mediators of Synapse Development and Plasticity.</a> Trends Immunol. 36 (10), 605-613 (2015).</li><li>Lou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptor+P2RY12-dependent+microglial+closure+of+the+injured+blood-brain+barrier.">Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier.</a> Proc Natl Acad Sci U S A. 113 (4), 1074-1079 (2016).</li><li>Haynes, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y12+receptor+regulates+microglial+activation+by+extracellular+nucleotides.">The P2Y12 receptor regulates microglial activation by extracellular nucleotides.</a> Nat Neurosci. 9 (12), 1512-1519 (2006).</li><li>Gosselin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), (2017).</li><li>Lavin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophage+enhancer+landscapes+are+shaped+by+the+local+microenvironment.">Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.</a> Cell. 159 (6), 1312-1326 (2014).</li><li>Hoeffel, G., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+Tissue-Resident+Macrophages.">Ontogeny of Tissue-Resident Macrophages.</a> Front Immunol. 6, 486 (2015).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci U S A. 113 (12), E1738-E1746 (2016).</li><li>Bohlen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Requirements+for+Microglial+Survival,+Specification,+and+Function+Revealed+by+Defined-Medium+Cultures.">Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.</a> Neuron. 94 (4), 759-773 (2017).</li><li>Herz, J., Filiano, A. J., Smith, A., Yogev, N., Kipnis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+Cells+in+the+Central+Nervous+System.">Myeloid Cells in the Central Nervous System.</a> Immunity. 46 (6), 943-956 (2017).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Because microglia serve as sentinel immune cells of the CNS, they are highly responsive to environmental changes; therefore, great care is required to minimize inflammatory responses within the cells during their isolation and culture8. This is accomplished in this protocol through speed and temperature. Keeping the cells on ice or at 4 &#176;C whenever possible greatly reduces activation, so all centrifugation steps take place at 4 &#176;C, the animals are perfused with ice-cold perfusion buffer,...

As macrophages of the CNS parenchyma, microglia interact with a vast array of neuronal circuitry and glial signaling networks. They play vital roles in development and homeostasis of the brain through synaptic pruning, apoptotic cell clearance, and transient interactions with neuronal processes1,2. Microglia are early responders to neurological injuries, extending their long, thin processes to lesion sites to coordinate inflammatory responses and limit bleeding3,4. Changes in microglial morphology and function are ubiquitous in both acute and chronic CNS injuries, and microglia exhibit altered morphology, localization, and expression of inflammatory mediators in a diverse range of disease states1. Human genetic studies indicate that mutations that alter risk for neurodegenerative diseases are often predominantly or exclusively expressed by microglia in the intact CNS, pointing to a critical role for microglia in disease pathogenesis or progression5. Given their prominence in injury and disease, furthering the understanding of microglial biology is a high priority for developing new therapeutic approaches.
Many critical advances to the understanding of microglial biology have arisen by extrapolating techniques and mechanisms discovered in studies of other macrophage populations including culture methods, gene expression profiles, and definitions of functional/morphological states. Although generalized macrophage functions often play out in surprising ways within the CNS landscape, microglia are themselves highly specialized, exhibiting a ramified morphology and a unique gene expression signature that sets them apart from other tissue macrophages6. Microglia have a lineage that is distinct from most other tissue macrophages; they colonize the CNS during an early embryonic wave of primitive hematopoiesis and self-renew throughout life, independent of contributions from definitive hematopoiesis7. The fully mature gene expression signature of adult microglia is not achieved until the second postnatal week8. Environmental cues from the surrounding tissue play a major role in dictating tissue-specific macrophage features6, which in the CNS includes limited exposure to blood-borne factors granted by the blood-brain barrier9.
One obstacle to fully understanding microglial contributions to CNS homeostasis and disease is the difficulty of recapitulating the specialized properties of mature microglia seen in vivo with purified cells in vitro. Many methods have been developed to isolate and culture intact microglia, but most approaches rely on serum to support cell survival. We have shown that addition of serum, which is an inherently variable reagent containing a vast array of bioactive molecules, is particularly problematic when working with microglia because it promotes an amoeboid morphology, increased proliferation, and increased phagocytosis9 often seen in vivo when microglia are exposed to blood borne factors after the disruption of the blood-brain barrier. By these metrics, serum-exposed cells resemble microglia in injury or disease states, but such alterations are reduced when microglia are cultured in defined growth medium containing CSF-1 (or IL-34), TGF-&#946;, cholesterol, and selenite.
This protocol provides details for culturing juvenile rat microglia under serum-free conditions, related to recently published work9. This protocol has been streamlined for rats from postnatal day 21 - 30 (P21 - P30), but can be adapted to isolate microglia from rats and mice of any age, though yield and overall viability will vary depending on the species and age of the animal. Maximal yields and optimal viability is achieved when using slightly immature microglia (~P9), with yields and viability gradually tapering to somewhat lower levels in adult animals. Microglia can also be isolated from mice, but we have found that rat cells show significantly higher yields, viability, and complexity of ramified morphologies, when compared to mouse cells in serum-free cultures. Animals aged greater than P50 have not been evaluated with this protocol. This immunopanning isolation procedure has been optimized to minimize changes in microglial transcriptional profiles during isolation and to maximize downstream viability of the cells. Using these techniques and media formulations, high-viability primary cultures can be sustained for weeks. Microglia cultured under these conditions exhibit a highly ramified morphology involving rapid extension and retraction of processes and relatively low rates of proliferation. We highlight the significance of serum-exposure on these properties, and discuss strengths and weakness of this method relative to other approaches.

<table>
	<tbody>
		<tr>
			<td>Rat: Sprague-Dawley</td>
			<td>Charles River</td>
			<td>Cat# 400</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-rat CD11b monoclonal (clone OX42)</td>
			<td>Bio-Rad</td>
			<td>Cat# MCA275R</td>
			<td>Panning: 1:1,000; Staining: 1:500</td>
		</tr>
		<tr>
			<td>Goat polycolonal anti-Iba1&#160;</td>
			<td>Abcam</td>
			<td>Cat# AB5076</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-Ki67&#160;</td>
			<td>Abcam&#160;</td>
			<td>Cat# AB15580</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-mouse 594</td>
			<td>Invitrogen</td>
			<td>Cat# 11055</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-goat 488</td>
			<td>Invitrogen</td>
			<td>Cat# A-21203</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-Rabbit 647</td>
			<td>Invitrogen</td>
			<td>Cat# A-31573</td>
			<td>Staining: 1:500</td>
		</tr>
		<tr>
			<td>Triton-X (detergent in ICC staining)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 28313</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td>Galen Laboratory Supplies</td>
			<td>Cat# GAG-HS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma&#160;</td>
			<td>Cat# M3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from milk solids</td>
			<td>Sigma</td>
			<td>Cat# P6838</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;2</td>
			<td>Peprotech</td>
			<td>Cat# 100-35B</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine IL-34</td>
			<td>R&#38;D Systems</td>
			<td>Cat# 5195-ML/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>Cat# 700000P</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Corning&#160;</td>
			<td>Cat# 354233</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 90260</td>
			<td></td>
		</tr>
		<tr>
			<td>11(Z)Eicosadienoic (Gondoic) Acid</td>
			<td>Cayman Chemicals&#160;</td>
			<td>Cat# 20606</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM dye</td>
			<td>Invitrogen</td>
			<td>Cat# C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer-1</td>
			<td>Invitrogen</td>
			<td>Cat# E1169</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>Cat# DPRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>GE Healthcare</td>
			<td>Cat# 17-5445-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>Cat# 21041-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Gibco</td>
			<td>Cat# 15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>Cat# 25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>Cat# 16634</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin&#160;</td>
			<td>Sigma</td>
			<td>Cat# T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>Cat# S-5261</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Gibco</td>
			<td>Cat# 11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>Cat# 0100-20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine&#160;</td>
			<td>Sigma</td>
			<td>Cat# A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria Plates&#160;</td>
			<td>VWR</td>
			<td>Cat# 62406-456</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Stock reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 10 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 5 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2.5 mg/mL in H2O 
			Concentration used: 1:25,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:100 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>TGF-b2</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 2 mg/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:1,000 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>Ovine wool cholesterol</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1.5 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparan sulfate</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 1 mg/mL in H2O 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Oleic acid/Gondoic acid</td>
			<td></td>
			<td></td>
			<td>Reconstitution: Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol 
			Concentration used: 1:1,000 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 50 mg/mL in PBS 
			Concentration used: 1:100 
			Storage: -20&#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +) 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Douncing Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++ 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Panning Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 2 mg/mL of peptone from milk solids in DPBS++</td>
		</tr>
		<tr>
			<td>Microglia Growth Medium (MGM)</td>
			<td></td>
			<td></td>
			<td>Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite 
			Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr>
			<td>Collagen IV Coating</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing 2 &#956;g/mL collagen IV</td>
		</tr>
		<tr>
			<td>Myelin Seperation Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2 
			Comments: Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr>
			<td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid 
			Comments: Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.</td>
		</tr>
	</tbody>
</table>

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.

This protocol describes a method to culture high purity ramified microglia from juvenile rats. Similar results can be obtained using immature, perinatal and adult animals, as discussed below. Since cell isolations often include subtle nuances and many opportunities for cells to die, we used quality-control checkpoints to help determine success at various steps. We typically monitored cell suspensions using a hemocytometer at multiple steps in the isolation protocol (F...

Watch this Scientific Journal Video about Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium at JoVE.com

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

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-culture-rodent-microglia-to-promote-dynamic-ramified

Research

JoVE Journal

Neuroscience

15.8K Views.  Stanford University. 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.

Video: Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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

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

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

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

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

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.

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 ...

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 ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...