Name: rapid isolation of dorsal root ganglion macrophages
Uploaded: 2019-09-07T14:30:00
Description: Watch this Scientific Journal Video about Rapid Isolation of Dorsal Root Ganglion Macrophages at JoVE.com

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
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	<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 ...

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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 co...

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>a-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 expres...

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 ...

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Neuroscience

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