Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Overview

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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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, and 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 laminectomy on the mouse placed in 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 incision starting from the thoracic region down to the sacral region. Remove the skin with 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 it into the same 50 mL conical tube with a 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, and 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 the 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.

Materials

Name	Company	Catalog Number	Comments
AP20187	Clontech	635058
a-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