Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Summary

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Abstract

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Introduction

Efficient nerve conduction in the mammalian central nervous system (CNS) requires myelination of axons by oligodendrocytes. During development, oligodendrocytes arise from a pool of oligodendrocyte progenitor cells (OPCs) that are thought to migrate from the ventricular zones of the developing forebrain and neural tube¹. After migration OPCs differentiate into mature, myelinating oligodendrocytes that not only facilitate efficient impulse propagation, but also provide axons with trophic support². The adult CNS maintains an abundant population of OPCs that are dispersed throughout the gray and white matter comprising approximately 5-8% of all cell....

Protocol

Procedures involving animal subjects have been approved by Institutional Animal Care and Use Committee (IACUC) and Johns Hopkins School of Medicine.

1. Preparation of Assay Plates, Stock Solutions, and OPC Base Media

Note: The rat OPC base (Sato) media described here has been derived from previously published studies^12,13. Alternative media formulations may also be compatible with this procedure.

1. Resuspend poly-L-lysine (PLL) to a concentration of 10 µg/ml in sterile deionized water. Pipette 400 μl of the diluted PLL into each well of a black-walled, clear bottom 24 well tissue culture grade dish.
2. Coat tissue cu....

Results

A2B5-positive OPCs are isolated through positive cell selection using a magnetic column separation system. Prior to the isolation procedure, the whole brain is removed from rat pups that are between P5 and P7. Once the whole brain is successfully detached from the skull, the olfactory bulbs and cerebellum are removed using fine surgical forceps (Figure 1A). In order to isolate cerebral cortical tissue the hypothalamus, thalamus and midbrain are excised by careful dissecti.......

Discussion

The protocol presented here describes a fast and reliable method for isolating rat OPCs and quantifying in vitro oligodendrogenesis in response to experimental factors. Using positive selection, high yields of viable and pure OPCs are used directly for experimental purposes. This method streamlines the isolation, culturing, and differentiation steps into a 1 week time frame, and fixed cells can be conveniently stored at 4 °C for several weeks without any loss in assay sensitivity.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissection Materials
PBS without Ca2+ and Mg2+	Mediatech	21-040-CV	1x
10 cm Polystyrene Petri Dishes	Fisherbrand	0857512
Light Dissection Microscope	Motic	SM2-168
Dissociation Materials
Tube Rotator	Miltenyi Biotec	130-090-753	Dissociation Tube Rotator
Neural Tissue Dissociation Kit (P)	Miltenyi Biotec	130-092-628	Enzymatic Dissociation Kit
PBS with Ca2+ and Mg2+	Corning	20-030-CV	1x
40 μm Cell Strainer	Corning	352340
A2B5 Column Purification
Anti-A2B5 Microbeads	Miltenyi Biotec	130-097-864	Bead Bound A2B5 Antibody
LS Columns	Miltenyi Biotec	130-042-401	Magnetic Selection Columns
EDTA	Corning	46-034-Cl	2 mM
PBS with Ca2+ and Mg2+	Corning	20-030-CV	1x
Bovine Serum Albumin	Sigma-Aldrich	A9647	0.50%
Culture Materials
PDGF-AA	PeproTech Inc	100-13A	20 ng/ml
Dimethyl sulfoxide	Sigma-Aldrich	D2650	Hybri-Max 100 ml
Triiodothyronine	Sigma-Aldrich	T6397	45 nM
Clemastine fumarate salt	Sigma-Aldrich	SML0445-100MG	1 μM
Quetiapine hemifumarate salt	Sigma-Aldrich	Q3638-10MG	1 μM
N-acetyl cysteine	Sigma-Aldrich	A9165	5 μg/ml
Progesterone	Sigma-Aldrich	P8783	60 ng/ml
Poly-L-lysine	Sigma-Aldrich	P1524	10 μg/ml
Putrecine	Sigma-Aldrich	P7505	16 μg/ml
Sodium Selenite	Sigma-Aldrich	S5261	40 ng/ml
Hydrocortisone	Sigma-Aldrich	H0135	50 ng/ml
d-Biotin	Sigma-Aldrich	B4639	10 ng/ml
Insulin	Sigma-Aldrich	I6634	5 μg/ml
Apo-Transferrin	Sigma-Aldrich	T1147	100 μg/ml
Bovine Serum Albumin	Sigma-Aldrich	A4161	100 μg/ml
Trace Elements B	Corning	25-022-Cl	1x
B-27 Supplement	Life Technologies	17504044	1x
Penicillin/Streptomycin	Life Technologies	15140122	1x
DMEM	Life Technologies	11995-065	1x
24-well Black Visiplate with lid	Perkin Elmer	1450-605	Tissue culture grade
Immunocytochemistry and Quantification
PFA	Sigma-Aldrich	100-13A	4%
Triton X-100	Sigma-Aldrich	78787	0.10%
Normal Goat Serum	Jackson Immuno Research	005-000-121	5%
mouse monoclonal anti-MBP	Biolegend	SMI-99P	1:1,000 Dilution
rabbit polyclonal anti-Actin	Santa Cruz Biotecnology	SC-7210	1:200 Dilution
rabbit polyclonal anti-Olig2	Millipore	AB9610	1:1,000 Dilution
goat anti-mouse 680RD	Licor	926-68070	1:500 Dilution
goat anti-rabbit 800CW	Licor	926-32211	1:500 Dilution
Alexa Fluor 594 goat anti-mouse	Life Technologies	A11032	1:1,000 dilution
Alexa Fluor 488 goat anti-rabbit	Life Technologies	A11008	1:1,000 dilution
Prolong Gold antifade with DAPI	Life Technologies	P36931
DPBS with Ca2+ and Mg2+	Corning	21-030-CV
Licor Odyssey Clx Infared Imaging System	Licor