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 contribute to neuropathic pain development after peripheral nerve injury by interacting with sensory neurons in the DRG^{5,6,7,8,9,10,11}. Moreover, recent studies also implicate the contribution of DRG macrophages in the axonal repair after nerve injury^12,13. Another study further suggests that macrophage subpopulations (i.e., CD11b⁺Ly6C^hi and CD11b⁺Ly6C^low/- 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 digestion^15,16. 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 °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.

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 pinch.
   NOTE: Both male and female mice aged 6-8 weeks were used.
3. Perfuse the mouse transcardially with 10 mL of pre-chilled 1x PBS.
   1. 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.
   2. Continue to use scissors to cut the diaphragm and the rib cage, open the pleural cavity to expose the beating heart.
   3. 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.
4. 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.
   1. 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.
   2. Use Friedman-Pearson Rongeur to peel off the connective tissues and muscles until the lumbosacral spine processes and bilateral transverse processes are exposed.
   3. 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.
5. 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.

2. Isolate single cells from mouse lumbar DRG

1. Homogenize the DRG tissue with a loose pestle in the Dounce homogenizer 20-25 times.
2. Place a sterile 70 μm nylon cell strainer in a sterile 50 mL conical tube. Wet the cell strainer with 800 μL of ice-cold 1x HBSS, and the flow-through is collected in the conical tube.
3. Collect the homogenized tissue suspension from the homogenizer using a pipette and pass through the wet 70 μm nylon cell strainer into the 50 mL conical tube.
4. Rinse the homogenizer twice with 800 μL of ice-cold 1x HBSS and then decant into the same 50 mL conical tube with cell strainer to increase the yield.
5. 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.
6. 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 μL of 1x HBSS to seal the top.
7. Pellet the cells by centrifugation at 800 x g for 20 min at 4 °C.
8. Carefully aspirate the supernatant containing myelin in the medium without disturbing the cell pellet at the bottom of the FACS tube.
9. 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.
   1. Resuspend the mechanically isolated DRG cells (L4/L5) in 100 μL of PBS containing 5% Fetal Bovine Serum and then incubate with α-mouse CX3CR1-APC antibody (1:2,000) in the dark at 4 °C for 1 h.
   2. Wash the cells with 5 mL of PBS once; centrifuge the cells 360 x g for 8 min at 4 °C.
   3. Aspirate the supernatant, then resuspend the cell pellet in 300 μL of PBS for FCAS analysis. If cell sorting is planned, resuspend the cells in the FACS buffer instead.

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