Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Summary

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Abstract

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Introduction

RGCs are terminally differentiated neurons, and therefore, primary cells are required for experimentation. The development of a protocol for the isolation and enrichment of primary murine retinal ganglion cells (RGCs) is fundamental to revealing the mechanisms of RGC health and degeneration in vitro. This is especially important for studies that seek to generate potential therapies to promote RGC function and to minimize their death. The degeneration of RGCs is associated with retinal degenerative diseases, such as glaucoma, diabetic retinopathy, and normal aging. Although the specific cellular mechanisms underlying RGC loss are unclear, a series of risk fact....

Protocol

All procedures detailed in the following protocol were approved by the Institutional Animal Care and Use Committee (IACUC) review board at the University of Tennessee Health Science Center (UTHSC) and followed the Association for Research in Vision and Ophthalmology (ARVO) Statements for the Use of Animals in Ophthalmic and Vision Research, in addition to the guidelines for laboratory animal experiments (Institute of Laboratory Animal Resources, Public Health Service Policy on Humane Care and Use of Laboratory Animals).

1. Preparation of Instruments, Solutions, and Media

Note: All information about....

Results

The in-depth study of RGCs is impeded by many factors, including their low frequency and the lack of a robust and standardized methodology for their isolation. Figure 1 shows the methodology used for retinae isolation. Variations in the enucleation procedure exist based on the type of analysis, such as if the enucleation is part of in vivo experimentation²⁷. Enucleation in this protocol is performed on euthanized mice. As show.......

Discussion

FACS is the technique of choice to purify cell populations. Other isolation methods include immunopanning, magnetic beads, and complement fixation depletion. The advantage of FACS over these other methodologies is based on the simultaneous identification of cell-surface markers with varying degrees of intensity. The fluorescent intensity of the molecule is proportional to the amount of protein expression. Until now, the isolation of RGCs was based solely on Thy1 (CD90) positivity and CD48 negativity¹⁵

Materials

Name	Company	Catalog Number	Comments
Anti-mouse CD15 PE	BioLegend	125606	Clone MC-480
Anti-mouse CD48 PE-Cy7	BioLegend	103424	Clone HM48-1
Anti-mouse CD57	Sigma Aldrich	C6680-100TST	Clone VC1.1
Anti-mouse CD90.2 AF700	BioLegend	105320	Clone 30-H12
Brilliant Violet 421 Goat Anti-mouse IgG	BioLegend	405317	Clone Poly4053
Purified Anti-mouse CD16/32	BioLegend	101302	FcgRII/III block, Clone 93
Zombie Aqua	BioLegend	423102	Live cell/ Dead cell discrimination
Fetal Bovine Serum	Hyclone	SH30071.03	U.S. origin
AbC Total Antibody Compensation Bead Kit	Thermo Fisher Scientific	A10497	Multi-species Ig
Neurobasal Medium	Thermo Fisher Scientific	21103049	Add serum to media prior to culture.
Phosphate-Buffered Saline (PBS)	Thermo Fisher Scientific	10010049	Saline solution
Dissection Microscope	Olympus	SZ-PT Model	Stereo Microscope
Sorvall Centrifuge	Thermo Scientific	ST 16R	All centrifugation performed  at RT
Base Plate – Dissection Pan	Fisher Scientific	SB15233FIM	A wax plate can also be used
Forceps	Aesculap	5002-7	4 ½ inches
Iris Scissors, Straight	Aesculap	1360	5 ½ inches
Falcon 15 mL conical tubes	Fisher Scientific	352097	Polypropylene tubes
Falcon 50 mL conical tubes	Fisher Scientific	352098	Polypropylene tubes
BD FACS Tubes	Fisher Scientific	352003	Polypropylene tubes
40 mm dishes	MidSci	TP93040	Tissue culture treated
70 μm nylon strainer	MidSci	70ICS	sterile
40 μm nylon strainer	MidSci	40ICS	sterile
BD 10 mL syringe	Fisher Scientific	301604	Disposable Syringe without needle
Pestles	MidSci	PEST	sterile
Wheaton Vials	Fisher Scientific	986734	No Liner
BD 30 G needle	Fisher Scientific	305128	1 inch
Hausser Scientific Bright-Line Glass Counting Chamber	Fisher Scientific	0267151B	Hemocytometer
Gibco Trypan blue 0.4% Solution	Fisher Scientific	15250061	Viability Dye
Eppendorf tubes	Fisher Scientific	05-402-25	1.5mL
EVOS Floid Cell Imaging	Thermo Fisher Scientific	447113	Fluorescence Imaging with a 20x objective
100% Ethanol	Fisher Scientific	04-355-452	Used to make 70% Ethanol
Pipet-Lite LTS Pipette L-1000XLS+	Rainin	17014282	LTS Pipette
Pipet-Lite LTS Pipette L-200XLS+	Rainin	17014391	LTS Pipette
Pipet-Lite LTS Pipette L-20XLS+	Rainin	17014392	LTS Pipette
Rack LTS 1000 mL – GPS-L1000S	Rainin	17005088	Blue Rack Sterile Tips
Rack LTS 250 mL – GPS-L250S	Rainin	17005092	Green Rack Sterile Tips
Rack LTS 20 mL – GPS-L10S	Rainin	17005090	Red Rack Sterile Tips
FACSAria II Cell Sorter	BD Biosciences	N/A	Custom order
LSR II Cytometer	BD Biosciences	N/A	Custom order
Abca8a	Thermo Fisher Scientific	Mm00462440_m1	Müller cells
Aldh1al	Thermo Fisher Scientific	Mm00657317_m1	Müller cells
Aqp4	Thermo Fisher Scientific	Mm00802131_m1	Astrocytes
Calb2	Thermo Fisher Scientific	Mm00801461_m1	Amacrine, Horizontal
Cd68	Thermo Fisher Scientific	Mm03047340_m1	Retinal Pigment Epithelial Cells
Gad2	Thermo Fisher Scientific	Mm00484623_m1	Amacrine
Hprt	Thermo Fisher Scientific	Mm01545399_m1	House keeping gene
Lhx1	Thermo Fisher Scientific	Mm01297482_m1	Horizontal
Lim2	Thermo Fisher Scientific	Mm00624623_m1	Horizontal
Nrl	Thermo Fisher Scientific	Mm00476550_m1	Photoreceptors
Ntrk1	Thermo Fisher Scientific	Mm01219406_m1	Horizontal
Pcp4	Thermo Fisher Scientific	Mm00500973_m1	Bipolar, Amacrine
Pou4f1	Thermo Fisher Scientific	Mm02343791_m1	Retinal Ganglion Cells
Prdx6	Thermo Fisher Scientific	Mm00725435_s1	Astrocytes
Prkca	Thermo Fisher Scientific	Mm00440858_m1	Bipolar
Prox1	Thermo Fisher Scientific	Mm00435969_m1	Horizontal
Pvalb	Thermo Fisher Scientific	Mm00443100_m1	Amacrine
Rbpms	Thermo Fisher Scientific	Mm02343791_m1	Retinal Ganglion Cells
Rom1	Thermo Fisher Scientific	Mm00436364_g1	Photoreceptors
Rpe65	Thermo Fisher Scientific	Mm00504133_m1	Retinal Pigment Epithelial cells
Slc1a3	Thermo Fisher Scientific	Mm00600697_m1	Astrocytes
Slc6a9	Thermo Fisher Scientific	Mm00433662_m1	Amacrine
Sncg	Thermo Fisher Scientific	Mm00488345_m1	Retinal Ganglion Cells
Tubb3	Thermo Fisher Scientific	Mm00727586_s1	Retinal Ganglion Cells
Vim	Thermo Fisher Scientific	Mm01333430_m1	Müller cells
Taqman Universal Master Mix	Thermo Fisher Scientific	4440047	qPCR Reagent
miRNeasy Mini Kit	Qiagen	217004	RNA Isolation
SuperScript VILO cDNA Synthesis Kit	Thermo Fisher Scientific	11754250	cDNA synthesis
Taqman PreAmp Master Mix	Thermo Fisher Scientific	4391128	Pre-Amplification step
BD Cytofix/ Cytoperm	BD Biosciences	554714	Fixation/ Permeabilization Buffer
BD Perm/ Wash	BD Biosciences	554723	Permeabilization Solution
RBPMS	Santa Cruz Biotechnology	sc-86815	intracellular antibody
SNCG	Gene Tex	GTX110483	intracellular antibody
BRN3A	Santa Cruz Biotechnology	sc-8429	intracellular antibody
TUJ1	BioLegend	801202	intracellular antibody