Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

Summary

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

Abstract

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

Introduction

The innate immune system includes multiple cell types that stimulate complement activation and inflammation. Innate immune cells include natural killer (NK) cells, mast cells, basophils, eosinophils, neutrophils, and mononuclear phagocytes. Mononuclear phagocytes, which are composed of monocytes, macrophages, and dendritic cells, have been implicated in the pathophysiology of multiple ophthalmic conditions including uveitis, diabetic retinopathy, and age-related macular degeneration (AMD)¹. In this protocol, we will focus on the identification of mononuclear phagocytes using multi-parameter flow cytometric analysis in a mouse model of neovascul....

Protocol

All procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. C57BL/6 mice were group housed at a barrier facility at the Center for Comparative Medicine at Northwestern University (Chicago, IL). All animals were housed in 12/12 h light/dark cycle with free access to food and water.

1. Collection of ocular tissue

1. Sacrifice 10-12-week-old C57BL/6J mice of either sex using regulated administration of carbon dioxide until mouse is unresponsiv.......

Discussion

Multi-parameter flow cytometry allows for the quantitative analysis of multiple cell types in a complex tissue. In this report, we describe the flow cytometric detection of mononuclear phagocyte populations, including monocytes, dendritic cells, and macrophage subsets, after laser injury in the mouse eye. Liyanage et al recently reported a similar gating strategy to detect neutrophils, eosinophils, lymphocytes, DCs, macrophages, infiltrating macrophages, and monocytes²⁷. Our gating strategy primar.......

Materials

Name	Company	Catalog Number	Comments
0.6 ml microcentrifuge tubes	Fisher Scientific	05-408-120
1.7 ml microcentrifuge tubes	Costar	3620
10 ml pipettes	Fisher Scientific	431031
15 ml conicals	ThermoFisher	339650
1x HBSS, 500 ml	Gibco	14025-092
2.5 ml syringe	Henke Sass Wolf	7886-1
20% paraformaldehyde solution	Electron Microscopy Sciences	15713-S
25 ml pipettes	Fisher Scientific	431032
30 G needle	Exel	26437
3ml luer lock syringe	Fisher Scientific	14955457
41 x 41 x 8 mm polystyrene weigh dish	Fisher Scientific	08-732-112
50 ml conicals	Falcon	352070
96-well, U-bottom assay plate without lid	Falcon	353910
Amine reactive beads	ThermoFisher	A10628
Analysis software	FlowJo	v 10
Anti-rat and anti-hamster Ig kappa bead set	BD Biosciences	552845
C57BL/6J mice	Jackson Laboratory	664
Cell counter	Invitrogen	AMQAX1000
Cell counter slides	Invitrogen	C10283
Compensation beads	ThermoFisher	01-1111-42
Count beads	ThermoFisher	01-1234-42
Curved forceps	Fisher Scientific	16-100-123
Digestion enzyme	Sigma Aldrich	5401020001
Dissecting microscope	National Optical and Scientific Instruments, Inc.	DC3-420T
Dissociation tubes	Miltenyi Biotec	130-096-334
DNase	Roche	10104159001
Electronic dissociator	Miltenyi Biotec	130-095-937
FACS Diva software	BD Biosciences
Fc block, rat anti-mouse CD16/CD32	BD Biosciences	553142
Fine forceps	Integra Miltex	17035X
Flow buffer	Miltenyi Biotec	130-091-221
Flow cytometer	BD Biosciences
Flow tubes	Falcon	352008
Hamster anti-mouse CD11c BV421	BD Biosciences	562782
Ice bucket	Fisher Scientific	07-210-123
Live/Dead dye	ThermoFisher	65-0866-14
Lysing solution, 10x solution	BD Biosciences	555899
Micro titer tube and rack	Fisher Scientific	02-681-380
Mouse anti-mouse CD64 PE	BioLegend	139304
Mouse anti-mouse NK1.1 PECF594	BD Biosciences	562864
P1000 pipet tips	Denville Scientific	P2404
P1000 pipettor	Gilson	FA10006M
P2 pipet tips	eppendorf	22491806
P2 pipettor	Gilson	FA10001M
P20 pipettor	Gilson	FA10003M
P200 pipet tips	Denville Scientific	P2401
P200 pipettor	Gilson	FA10005M
Pipet man	Fisher Scientific	FB14955202
Rat anti-mouse B220 PECF594	BD Biosciences	562313
Rat anti-mouse CD11b APC-Cy7	BD Biosciences	557657
Rat anti-mouse CD19 AlexaFluor 700	BD Biosciences	557958
Rat anti-mouse CD19 PE	BD Biosciences	553786
Rat anti-mouse CD4 PECF594	BD Biosciences	562314
Rat anti-mouse CD45 FITC	ThermoFisher	11-0451-82
Rat anti-mouse CD45 PE-Cy7	BD Biosciences	552848
Rat anti-mouse CD8 PECF594	BD Biosciences	562315
Rat anti-mouse Ly6C	BD Biosciences	561085
Rat anti-mouse Ly6G PECF594	BD Biosciences	562700
Rat anti-mouse MHC II AlexaFluor 700	BioLegend	107622
Rat anti-mouse Siglec F PECF594	BD Biosciences	562757
Rat anti-mouse Tim4 AlexaFluor647	BD Biosciences	564178
Shaking incubator	Labnet	311DS
Spring scissors	Fine Science Tools	15024-10
Sterile cell strainer, 40 mm nylon mesh	Fisher Scientific	22363547
Sterile water, 500 ml	Gibco	A12873-01
Swinging bucket centrifuge	eppendorf	5910 R
Trypan blue, 0.4% solution	Gibco	15250061