In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Summary

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Abstract

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

Introduction

Bone marrow-derived macrophages (BMDMs) are an important link between the innate and adaptive immune systems. As antigen presenting cells (APCs), they can communicate with lymphocytes via both antigen presentation and cytokine release^1,2,3. However, as professional phagocytes, their primary function is to clear pathogens, aged cells, and cellular debris^1,4. Following a spinal cord injury (SCI), substantial quantities of myelin debris is generated from dying oligodendrocytes, the cell type responsible for CNS axon myel....

Protocol

The methods described here and in Section 2 have been approved by the Florida State University Institutional Animal Care and Use Committee (IACUC) and follows the guidelines set forth in the Guide for Care and Use of Laboratory Animals, 8^th edition. All animals used in this in this protocol are house in a dedicated laboratory animal facility until use. No in vivo experimentation was performed prior to sacrifice. Animal numbers were based on experimental need using average cell and myelin collections as a guide in order to minimize usage.

NOTE: This protocol describes the generation of bone marro....

Results

Treatment of BMDMs with CFSE labeled myelin debris should yield clear internalization (Figure 2). While a 3-hour interaction time is sufficient for BMDMs to phagocytose enough added myelin debris for robust downstream detection, intracellular accumulation can be observed with as little as 1 hour of interaction. However, some myelin debris may still be present on the cell surface after washing. This may be due to insufficient washing, or particles not being fully internalized during the early.......

Discussion

The procedures described here utilize both freshly isolated crude CNS myelin debris and primary bone marrow-derived macrophages. To reduce animal expenditures, we recommend that both brains and bone marrow cells be harvested from each mouse at the time of sacrifice. Two researchers working together can prepare both materials simultaneously. Alternatively, brains can be stored at -80 °C in PBS supplemented with antibiotics prior to myelin debris isolation. It has been our experience that brains can be maintained in t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	GE Healthcare Life Sciences	SH30243.01	High glucose with L-glutamine, sodium pyruvate
Penicillin-Streptomycin Solution	Corning	30-002-CI	100X Solution
New Born Calf Serum (NCS)	Rocky Mountain Biologics	NBS-BBT-5XM	United States Origin
NCTC clone 929 [L cell, L-929, derivative of Strain L]	ATCC	CCL-1	L929 Cell Line of Conditioned Media Preparation
24-well Cell Culture Plates	VWR	10062-896
Cell Culture Dish	Greiner Bio-One	639960	Polystyrine, 145/20mm
CFSE Cell Proliferation Kit	Thermo Fisher	C34570	DMSO for Reconsitution Provided
Fluoro-gel with Tris Buffer	Electron Microscopy Sciences	17985-11
Oil Red O	Sigma Aldrich	O0625
Equipment
Materials	Company	Catalog Number	Comments
Ultracentrifuge Tubes	Beckman Coulter	326823	Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm
SW 32 Ti Ultracentrifuge Rotor	Beckman Coulter	369650	SW 32 Ti Rotor, Swinging Bucket, Titanium
Hand Held Rotary Homogenizer	Fisher Science	08-451-71