The study was supported by: Foundation for Anesthesia Education and Research (XY); the UCSF Department of Anesthesia and Perioperative Care (XY); and 1R01NS100801-01(GZ). This study was supported in part by HDFCCC Laboratory for Cell Analysis Shared Resources Facility through a grant from the NIH (P30CA082103).

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
1. Collect lumbar DRG from experimental mice
<ol>
	<li>Before starting the experiment, prepare the working solution of the density gradient medium (e.g., Percoll) by mixing 9 volumes of the medium with 1 volume of Ca++/Mg++-free 10x HBSS. Keep it on ice.</li>
	<li>Anesthetize the mouse with 2.5% Avertin. Confirm that the animal is fully anesthetized by the lack of response to the hind paw pinch. 
	NOTE: Both male and female mice aged 6-8 weeks were used.</li>
	<li>Perfuse the mouse transcardially with 10 mL of pre-chilled 1x PBS.
	<ol>
		<li>Place the mouse in the supine position with four paws secured with the tape inside a chemical fume hood. Lift the skin below the rib cage by the forceps, and make a small incision with surgical scissors to expose the liver and diaphragm.</li>
		<li>Continue to use scissors to cut the diaphragm and the rib cage, open the pleural cavity to expose the beating heart.</li>
		<li>Quickly cut the right atrial appendage with iris scissors. Once the bleeding is noted, insert a 30 G needle into the posterior end of the left ventricle, and slowly inject 10 mL of pre-chilled 1x PBS to perfuse the animal.</li>
	</ol>
	</li>
	<li>Perform dorsal laminectomy15 on the mouse placed at the prone position. 
	NOTE: If cell culture is planned, spray the mouse with 70% ethanol before incision and use pre-sterilized surgical instruments for dissection.
	<ol>
		<li>Use a size 11 scalpel to make one longitudinal lateral deep incisions starting from the thoracic region down to the sacral region. Remove the skin by the scissors to expose the dorsal muscle layer.</li>
		<li>Use Friedman-Pearson Rongeur to peel off the connective tissues and muscles until the lumbosacral spine processes and bilateral transverse processes are exposed.</li>
		<li>Use a Noyes Spring Scissor to carefully open the dorsal spinal column, then switch to a Friedman-Pearson Rongeur to remove the vertebral bones to expose the spinal cord with intact spinal nerves attached.</li>
	</ol>
	</li>
	<li>Carefully dissect ipsilateral and contralateral lumbar DRG (L4/L5 DRG in our study) and place it into 1 mL of ice-cold Ca++/Mg++-free 1x HBSS in a Dounce tissue homogenizer. Now the tissues are ready for step 2. 
	NOTE: Trim the spinal nerve attached to the DRG if possible.</li>
</ol>
2. Isolate single cells from mouse lumbar DRG
<ol>
	<li>Homogenize the DRG tissue with a loose pestle in the Dounce homogenizer&#160;20-25 times.</li>
	<li>Place a sterile 70 &#956;m nylon cell strainer in a sterile 50 mL conical tube. Wet the cell strainer with 800 &#956;L of ice-cold 1x HBSS, and the flow-through is collected in the conical tube.</li>
	<li>Collect the homogenized tissue suspension from the homogenizer using a pipette and pass through the wet 70 &#956;m nylon cell strainer into the 50 mL conical tube.</li>
	<li>Rinse the homogenizer twice with 800 &#956;L of ice-cold 1x HBSS and then decant into the same 50 mL conical tube with cell strainer to increase the yield.</li>
	<li>Add 1.5 mL of equilibrated ice-cold isotonic density gradient medium (prepared in step 1.1) into a sterile 5-mL polystyrene FACS tube.</li>
	<li>Transfer the cell homogenate from the 50 mL conical tube (in step 2.4) into the FACS tube, mix well with the density gradient medium by pipetting up and down. Add an additional 500 &#956;L of 1x HBSS to seal the top.</li>
	<li>Pellet the cells by centrifugation at 800 x g for 20 min at 4 &#176;C.</li>
	<li>Carefully aspirate the supernatant containing myelin in the medium without disturbing the cell pellet at the bottom of the FACS tube.</li>
	<li>Resuspend the cells in PBS or FACS buffer containing 5% Fetal bovine serum (FBS) for FACS analysis. 
	NOTE: At least 50,000 to 100,000 cells are expected from L4 /L5 DRG of one mouse.
	<ol>
		<li>Resuspend the mechanically isolated DRG cells (L4/L5) in 100 &#956;L of PBS containing 5% Fetal Bovine Serum and then incubate with &#945;-mouse CX3CR1-APC antibody (1:2,000) in the dark at 4 &#176;C for 1 h.</li>
		<li>Wash the cells with 5 mL of PBS once; centrifuge the cells 360 x g for 8 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant, then resuspend the cell pellet in 300 &#956;L of PBS for FCAS analysis. If cell sorting is planned, resuspend the cells in the FACS buffer instead.</li>
	</ol>
	</li>
</ol>

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<li>Zhang, H., Li, Y., de Carvalho-Barbosa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dorsal+Root+Ganglion+Infiltration+by+Macrophages+Contributes+to+Paclitaxel+Chemotherapy-Induced+Peripheral+Neuropathy%5BTitle%5D)+AND+%22The+Journal+of+Pain%22%5BJournal%5D)">Dorsal Root Ganglion Infiltration by Macrophages Contributes to Paclitaxel Chemotherapy-Induced Peripheral Neuropathy.</a> The Journal of Pain. 17 (7), 775-786 (2016).</li>
<li>Liu, T., van Rooijen, N., Tracey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depletion+of+macrophages+reduces+axonal+degeneration+and+hyperalgesia+following+nerve+injury%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Depletion of macrophages reduces axonal degeneration and hyperalgesia following nerve injury.</a> Pain. 86 (1-2), 25-32 (2000).</li>
<li>Peng, J., Gu, N., Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+and+monocytes+synergistically+promote+the+transition+from+acute+to+chronic+pain+after+nerve+injury%5BTitle%5D)+AND+%22Nature+Communication%22%5BJournal%5D)">Microglia and monocytes synergistically promote the transition from acute to chronic pain after nerve injury.</a> Nature Communication. 7, 12029(2016).</li>
<li>Barclay, J., Clark, A. K., Ganju, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+cysteine+protease+cathepsin+S+in+neuropathic+hyperalgesia%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Role of the cysteine protease cathepsin S in neuropathic hyperalgesia.</a> Pain. 130 (3), 225-234 (2007).</li>
<li>Lim, H., Lee, H., Noh, K., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IKK%2FNF-kappaB-dependent+satellite+glia+activation+induces+spinal+cord+microglia+activation+and+neuropathic+pain+after+nerve+injury%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">IKK/NF-kappaB-dependent satellite glia activation induces spinal cord microglia activation and neuropathic pain after nerve injury.</a> Pain. 158 (9), 1666-1677 (2017).</li>
<li>Rutkowski, M. D., Pahl, J. L., Sweitzer, S., van Rooijen, N., DeLeo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limited+role+of+macrophages+in+generation+of+nerve+injury-induced+mechanical+allodynia%5BTitle%5D)+AND+%22Physiology+and+Behavior%22%5BJournal%5D)">Limited role of macrophages in generation of nerve injury-induced mechanical allodynia.</a> Physiology and Behavior. (3-4), 225-235 (2000).</li>
<li>Kwon, M. J., Shin, H. Y., Cui, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CCL2+Mediates+Neuron-Macrophage+Interactions+to+Drive+Proregenerative+Macrophage+Activation+Following+Preconditioning+Injury%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.</a> Journal of Neuroscience. 35 (48), 15934-15947 (2015).</li>
<li>Zigmond, R. E., Echevarria, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+biology+in+the+peripheral+nervous+system+after+injury%5BTitle%5D)+AND+%22Progress+in+Neurobiology%22%5BJournal%5D)">Macrophage biology in the peripheral nervous system after injury.</a> Progress in Neurobiology. 173, 102-121 (2019).</li>
<li>Ghasemlou, N., Chiu, I. M., Julien, J. P., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD11b%2BLy6G-+myeloid+cells+mediate+mechanical+inflammatory+pain+hypersensitivity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+USA%22%5BJournal%5D)">CD11b+Ly6G- myeloid cells mediate mechanical inflammatory pain hypersensitivity.</a> Proceedings of the National Academy of Science USA. 112 (49), E6808-E6817 (2015).</li>
<li>Malin, S. A., Davis, B. M., Molliver, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+dissociated+sensory+neuron+cultures+and+considerations+for+their+use+in+studying+neuronal+function+and+plasticity%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.</a> Nature Protocols. 2 (1), 152-160 (2007).</li>
<li>Lin, Y. T., Chen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dorsal+Root+Ganglia+Isolation+and+Primary+Culture+to+Study+Neurotransmitter+Release%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiment%22%5BJournal%5D)">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> Journal of Visualized Experiment. (140), (2018).</li>
<li>Burnett, S. H., Kershen, E. J., Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+macrophage+ablation+in+transgenic+mice+expressing+a+Fas-based+suicide+gene%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene.</a> J Leukoc Biol. 75 (4), 612-623 (2004).</li>
<li>Lopes, D. M., Malek, N., Edye, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+peripheral+not+central+immune+responses+to+pain-inducing+injury%5BTitle%5D)+AND+%22Science+Reports%22%5BJournal%5D)">Sex differences in peripheral not central immune responses to pain-inducing injury.</a> Science Reports. 7 (1), 16460(2017).</li>
<li>Kim, Y. S., Anderson, M., Park, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coupled+Activation+of+Primary+Sensory+Neurons+Contributes+to+Chronic+Pain%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain.</a> Neuron. 91 (5), 1085-1096 (2016).</li>
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The authors declare no competing financial interests related to this manuscript.

Here we introduce a new method to effectively enrich isolated macrophages from mouse DRG. The conventional approach to isolate DRG immune cells requires enzymatic digestion15,18, which is now replaced with mechanical homogenization in our protocol to limit undesired cell damage and increase the yield. Therefore, the new protocol is far less time consuming. More importantly, enzyme digestion might stimulate the macrophages and change the molecular signature. In contrast, our mechanical approach greatly limits cellular stress.
Using FACS analysis, we further characterized the macrophages in the isolated DRG cells and demonstrated a great cellular recovery. More than 80% of isolated DRG cells under this protocol were found to be viable (Figure 2D). At least 50,000 to 100,000 cells are expected from L4 /L5 DRG of one mouse. Therefore, we can confidently conclude that the DRG cells can be further sorted into macrophage subpopulations14 for either culture or RNA isolation. However, there are a few critical steps which may impact the yield. If the unsatisfying cell yield is noted, the insufficient or excessive homogenization in step 2.1 should be suspected. The extent of cell death assessed with PI (Figure 2) or 7-AAD staining can be utilized to optimize the protocol. In addition, DRG dissection in step 1.4 may require repeated practice for beginners.
Currently, our lab mainly uses this protocol for studying the DRG macrophages. Likely, the application of this protocol can be expanded to study other non-neuronal cells in the DRG, such as satellite cells19, and T cells20. Further studies are needed to confirm the effectiveness of cell isolation. Unfortunately, our current mechanical dissociation method is not ideal for isolation of DRG sensory neurons, and enzymatic digestion remains the most effective method for sensory neuron isolation15.

There is now considerable evidence that immune cells contribute to the neuropathic pain following peripheral nerve injury1,2. Peripheral monocytic cells, including mature macrophages, are known to respond to tissue injury and systemic infection through phagocytosis, antigen presentation, and cytokine release. Paralleling the nerve injury-induced microglia activation in the spinal dorsal horn, macrophages in the dorsal root ganglia (DRG) also expand significantly after nerve injury3,4. Notably, there are growing interests to determine if macrophages contribute to neuropathic pain development after peripheral nerve injury by interacting with sensory neurons in the DRG5,6,7,8,9,10,11. Moreover, recent studies also implicate the contribution of DRG macrophages in the axonal repair after nerve injury12,13. Another study further suggests that macrophage subpopulations (i.e., CD11b+Ly6Chi and CD11b+Ly6Clow/- cells) may play a different role in the mechanical hypersensitivity14. Therefore, rapidly phenotyping the response of DRG macrophages in the context of nerve injury may help us identify neuroimmune factors contributing to neuropathic pain.
Conventionally, the protocol to isolate macrophages in the DRG involves multiple steps including enzymatic digestion15,16. The technique is often time consuming and can be costly for large-scale experiments. Although mild digestion with collagenase type II (4 mg/mL) and dispase type II (4.7 mg/mL) for 20 min was recommended previously15, it is conceivable that cells after the exposure to this enzyme are prone to cell damage or cell death, which may lead to low yield. In addition, the difference in the quality of enzymes from batch to batch may further impact the efficiency of this process. More importantly, macrophages exposed to the enzyme digestion might be undesirably stimulated and thus can be very different from the in-vivo status. The changes may potentially complicate the outcome of the functional study.
Here we describe an enzyme-free protocol to rapidly isolate DRG macrophages at 4 &#176;C using mechanical dissociation. The samples are kept on ice to limit cellular stress. As a result, our approach provides an advantage to maintain consistency of the isolation, and the isolated cells are presumably healthier and less stimulated. We further present the evidence to validate the quality of the isolated cells with Fluorescence-activated Cell Sorting (FACS) analysis.

<table><tbody><tr><td>AP20187</td><td>Clontech</td><td>635058</td><td></td></tr><tr><td>&amp;#945;-mouse CX3CR1-APC antibody</td><td>Biolegend</td><td>149007</td><td></td></tr><tr><td>Avertin</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Cell strainer (70 mm nylon)</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Dounce tissue homogenizer</td><td>Wheaton</td><td>357538 (1ml)</td><td></td></tr><tr><td>FACS tubes (5ml)</td><td>Falcon</td><td>352052</td><td></td></tr><tr><td>Friedman-Pearson Rongeur</td><td>FST</td><td>16121-14</td><td></td></tr><tr><td>HBSS (10x, Ca++/Mg++-free)</td><td>Gibco</td><td>14185-052</td><td></td></tr><tr><td>Noyes Spring Scissor</td><td>FST</td><td>15012-12</td><td></td></tr><tr><td>Percoll</td><td>Sigma</td><td>P4937-500ml</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma</td><td>P4864-10ml</td><td></td></tr></tbody></table>

rapid isolation of dorsal root ganglion macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

To validate the isolated cells, we first chose the Macrophage Fas-Induced Apoptosis (MAFIA) transgenic mice17. This line expresses a drug-inducible FK506-binding protein (FKBP)-Fas suicide fusion gene and green fluorescent protein (eGFP) under the control of the promoter of CSF1 receptor (CSF1R), which is specifically expressed in both macrophages and microglia. Systemic injection of FK-binding protein dimerizer, AP20187 (AP), induces the apoptosis of the cells expressing the transgene. The expression of EGFP also allows us to monitor the macrophages in the DRG. To deplete macrophages in the MAFIA mice, we began our studies with 3 daily intraperitoneal injections of AP (1 mg/kg). After the last injection, DRG were sectioned for immunostaining for GFP. We recorded a significant loss of GFP+ cells in the DRG of the AP-treated mice compared to VEH-treated mice (Figure 1A-B). In a separate experiment, we used this protocol to mechanically dissociate the DRG macrophages after the treatment. Subsequent FACS analysis revealed a successful depletion of GFPhi population in AP-treated mice (Figure 1C-D) and demonstrated the high quality of isolated cells.
We also characterized the isolated DRG cells from the wild-type mice. Mechanically isolated DRG cells (L4/L5) were stained with &alpha;-mouse CX3CR1-APC antibody. We found that 6% of the DRG cells were CX3CR1+ macrophages (Figure 2A-B). Cell viability was also assessed with Propidium Iodide (final concentration of 2.5 &#956;g/ml) which binds to the intracellular DNA of the nonviable cells, revealing that more than 80% of freshly isolated DRG cells were viable (Figure 2C-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60023/60023fig01.jpg" /> 
Figure 1: FACS analysis of macrophages in the DRG of MAFIA mice after AP treatment. 
MAFIA mice received daily intraperitoneal injection of 1 mg/kg of AP20187 (AP) or vehicle (VEH) for 3 days before the analysis. (A, B) Representative immunostaining images showing the AP-induced depletion of GFP+ (green) macrophages in the L4/L5 DRG after the 3rd AP treatment. NF200 (blue) was used to label myelinated neurons. Scale bar: 50 &#956;m. (C, D) The percent CSF1R-GFPhi cells after mechanical dissociation of the L4/5 DRG was determined by FACS analysis, and a representative data set from three independent experiments is shown with the percentage of the gated cell population indicated. The result shows that 4% of total isolated cells from the DRG of VEH-treated animal were GFPhi macrophages. In contrast, only 0.4% of total DRG cells were GFPhi macrophages in AP-treated mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60023/60023fig01large.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/60023/60023fig02.jpg" /> 
Figure 2: FACS characterization of macrophages in the DRG of the wild-type mice. 
(A, B) Ipsilateral L4 and L5 DRG of na&#239;ve wild-type mouse were pooled for mechanical dissociation. The percent CX3CR1+ macrophages were measured by FACS analysis (A). The gating for CX3CR1+ cells was based on the background fluorescence in the cells incubated with APC-conjugated isotype control antibody (B). (C, D) Cell viability of freshly isolated DRG cells was assessed with Propidium Iodide (PI) staining. PI+ cells (C) were gated based on the background fluorescence in the unstained cells (D). A representative data set from three independent experiments is shown with the percentage of the gated cell population indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60023/60023fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid Isolation of Dorsal Root Ganglion Macrophages at JoVE.com

Rapid Isolation of Dorsal Root Ganglion Macrophages

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

rapid-isolation-of-dorsal-root-ganglion-macrophages

Nanyang Technological University

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10.2K Views.  University of California San Francisco. Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Video: Rapid Isolation of Dorsal Root Ganglion Macrophages

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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

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

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

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

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Extraction and Enzymatic Purification of Microglia from the Mouse Spinal Cord

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Author Spotlight: Microglia Research on Spinal Cord Heterogeneity and Purification 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Source: Yu, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60023">Rapid Isolation of Dorsal Root Ganglion Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate macrophages from mouse dorsal root ganglion (DRG) using a mechanical dissociation technique. Upon homogenization of the freshly collected DRG tissue, antibodies against the cell-surface receptors on the macrophages are used to label the cells fluorescently, followed by performing flow cytometry to confirm the presence of macrophages in the sample.

Source: Yu, X., et al. Rapid Isolation of Dorsal Root Ganglion Macrophages. J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

Source: Maguire, A. D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64603">Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.</a> J. Vis. Exp. (2023)
This video demonstrates a protocol for enriching neuronal cultures from dorsal root ganglia by immunopanning, thereby improving their purity and utility for neurological research.

1. Experimental method

	Day 1: preparation of dishes
	
		In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the ...

Neuronal Culture Techniques

Co-culture and Interaction Studies

A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

Source: de Luca, A. C., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52543">Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration.</a> J. Vis. Exp. (2015).
This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat&#39;s spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Set-up

	Prior to ...

Primary Neuronal Culture

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Source: Lin, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57569">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for isolating and culturing dorsal root ganglia (DRG) cells from the rat spinal column. DRGs excised from the spinal column are treated with enzymes and mechanically dissociated to obtain a single-cell suspension. The cells are cultured in a medium containing inhibitors and growth factors that limit glial cell proliferation and promote neuronal growth, respectively.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Collect Lumbar DRG from ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

Summary

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Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

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

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column