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

Steven Droho; Carla  M. Cuda; Jeremy  A. Lavine

doi:10.3791/61348

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

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

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

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09:58 min

June 17, 2020

DOI: 10.3791/61348-v

Steven Droho¹, Carla M. Cuda*², Jeremy A. Lavine*¹

¹Department of Ophthalmology, Feinberg School of Medicine,Northwestern University, ²Department of Medicine, Division of Rheumatology, Feinberg School of Medicine,Northwestern University

Overview

This protocol describes a method for digesting whole mouse eyes into a single cell suspension for multi-parameter flow cytometry analysis. It focuses on identifying specific ocular mononuclear phagocytic populations, including macrophages, monocytes, microglia, and dendritic cells.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

The technique allows for quantitative analysis using fluorophore intensity.
It helps differentiate closely-related cell types based on cell surface marker expression.
Optimized for macrophage heterogeneity analysis.
Applicable to various models such as diabetic retinopathy and autoimmune uveitis.

Purpose of Study

To analyze mononuclear phagocyte populations in ocular tissues.
To enhance understanding of macrophage roles in ocular diseases.
To provide a reliable method for flow cytometric analysis.

Methods Used

Digestion of whole mouse eyes into a single cell suspension.
Multi-parameter flow cytometry for cell analysis.
Use of fluorescently conjugated antibodies for cell surface marker detection.
Optimization based on cytometer configuration.

Main Results

Successful identification of distinct ocular mononuclear phagocyte populations.
Quantitative differentiation of macrophage subtypes.
Insights into the heterogeneity of macrophages in ocular models.
Protocol adaptable to various experimental conditions.

Conclusions

The protocol is effective for analyzing ocular immune cells.
It provides a framework for studying macrophage dynamics in eye diseases.
Understanding cytometer configuration is crucial for successful implementation.

Frequently Asked Questions

What is the main advantage of this protocol?

The main advantage is the ability to use quantitative analysis with fluorophore intensity to differentiate closely-related cell types.

Can this protocol be used for other models?

Yes, it can be applied to models like diabetic retinopathy and experimental autoimmune uveitis.

What is essential to consider when using this protocol?

Understanding your cytometer's configuration is critical as it affects antibody volumes and fluorophore adjustments.

What types of cells can be identified using this method?

The method identifies monocytes, microglia, macrophages, and dendritic cells.

Is this protocol optimized for any specific type of macrophage?

Yes, it is optimized for analyzing both resident and infiltrating macrophage populations.

What is the purpose of using fluorescently conjugated antibodies?

They are used to detect specific cell surface markers for accurate cell identification.

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.

This protocol is designed to digest whole mouse eyes into a single cell suspension for multi-parameter flow cytometry of mononuclear phagocytes which include macrophages, monocytes, microglia, and dendritic cells. The main advantage of this technique is the ability to use quantitative analysis with fluorophore intensity to differentiate closely-related cell types using cell surface marker expression. Our protocol is optimized for the analysis of macrophage heterogeneity, including both resident and infiltrating populations in the laser-induced choroidal neovascularization model.

However, these concepts and techniques can be equally applied to other models like diabetic retinopathy, experimental autoimmune uveitis, as well as manifestations of lupus. When attempting this protocol, understanding your cytometer's configuration is a critical starting point as all cytometers are not configured the exact same way. Thus, volumes of fluorescently conjugated antibodies may need to be titrated and/or fluorophores may need to be adjusted to optimize the protocol based on your cytometer's configuration.

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