Rapid Isolation of Dorsal Root Ganglion Macrophages

Summary

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

Abstract

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 “rapid isolation of”) 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.

Introduction

There is now considerable evidence that immune cells contribute to the neuropathic pain following peripheral nerve injury^1,2. 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 injury^3,4. Notably, there are growing interests to determine if macrophages con....

Protocol

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

1. 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.
2. Anesthetize the mouse with 2.5% Avertin. Confirm that the animal is fully anesthetized by the lack of response to the hind paw pi....

Results

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.......

Discussion

Here we introduce a new method to effectively enrich isolated macrophages from mouse DRG. The conventional approach to isolate DRG immune cells requires enzymatic digestion^15,18, 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AP20187	Clontech	635058
α-mouse CX3CR1-APC antibody	Biolegend	149007
Avertin	Sigma	T48402
Cell strainer (70 mm nylon)	Falcon	352350
Centrifuge	Eppendorf	5810R
Dounce tissue homogenizer	Wheaton	357538 (1ml)
FACS tubes (5ml)	Falcon	352052
Friedman-Pearson Rongeur	FST	16121-14
HBSS (10x, Ca++/Mg++-free)	Gibco	14185-052
Noyes Spring Scissor	FST	15012-12
Percoll	Sigma	P4937-500ml
Propidium iodide	Sigma	P4864-10ml