Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Aymric Kisserli; Sandra Audonnet; Val&#233;rie Duret; Thierry Tabary; Jacques Henri Max Cohen; Rachid Mahmoudi

doi:10.3791/60810

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

JoVE Journal
Immunology and Infection

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Immunology and Infection

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Full Text

7,777 Views

07:20 min

May 19, 2020

DOI: 10.3791/60810-v

Aymric Kisserli^1,2, Sandra Audonnet³, Valérie Duret^2,4, Thierry Tabary^2,4, Jacques Henri Max Cohen², Rachid Mahmoudi^5,6

¹Oncogeriatric Coordination Unit,Reims University Hospitals, Maison Blanche Hospital, ²Faculty of Medicine,University of Reims Champagne-Ardenne, ³URCACyt, Flow cytometry technical platform,University of Reims Champagne-Ardenne, ⁴Department of Immunology,Reims University Hospitals, Robert Debre Hospital, ⁵Department of Internal Medicine and Geriatrics,Reims University Hospitals, Maison Blanche Hospital, ⁶Faculty of Medicine,University of Reims Champagne-Ardenne

Overview

This protocol outlines a method to measure CR1 density in erythrocytes using flow cytometry and immunostaining. It is particularly useful for evaluating receptor expression in various conditions such as Alzheimer's and systemic lupus erythematosus.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

CR1 is a receptor involved in immune response.
Changes in CR1 density can indicate various diseases.
Flow cytometry allows for precise measurement of receptor density.
Immunostaining enhances the detection of specific receptors.

Purpose of Study

To determine CR1 density in erythrocytes of subjects.
To compare CR1 density with known values from other subjects.
To assess the impact of diseases on CR1 expression.

Methods Used

Flow cytometry for quantifying receptor density.
Immunostaining with anti-CR1 monoclonal antibody.
Use of phycoerythrin (PE) for enhanced detection.
Comparison with control subjects to evaluate results.

Main Results

Robust results for low-density receptors.
Evaluation of CR1 expression reduction in various diseases.
Visual demonstration aids in understanding analysis parameters.
Protocol applicable for fluorescence microscopy studies.

Conclusions

The method is effective for measuring CR1 density.
It provides insights into receptor expression in health and disease.
Collaboration with cytometric specialists is recommended.

Frequently Asked Questions

What is CR1?

CR1 is a receptor involved in the immune response, particularly in the clearance of immune complexes.

How does flow cytometry work?

Flow cytometry analyzes the physical and chemical characteristics of cells or particles as they flow in a fluid stream through a beam of light.

Why is immunostaining used?

Immunostaining is used to detect specific proteins in cells or tissues using antibodies, enhancing visualization and analysis.

What diseases can affect CR1 density?

Diseases such as Alzheimer's, systemic lupus erythematosus, AIDS, and malaria can affect CR1 density.

Is this method applicable to other receptors?

Yes, this protocol can be adapted for analyzing other cell receptors as well.

What is the role of phycoerythrin in this method?

Phycoerythrin is a fluorescent dye that enhances the detection of the antibody-bound receptors during flow cytometry.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects' erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

This protocol can be used to measure the number of antigenic sites on cellular receptor of interest. The main advantage of this technique is that it produces robust results, even for receptors expressed at a low density. This method enables the evaluation of the reduction of CR1 erythrocyte expression and this is such as Alzheimer's, systemic lupus erythematosus, AIDS, and malaria.

This protocol is useful for any analysis of cell receptor density and can also be applied to the study of cell receptor expression by fluorescence microscope. We recommend spacing out the tubes during the cell and antibody distribution, and to be accompanied by a cytometric specialist during the first analysis if possible. Visual demonstration of the immunostaining and density quantification by flow cytometry is critical for understanding how to properly set the analysis parameters.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Erythrocyte Complement Receptor 1 Flow Cytometry Antigenic Sites Cell Receptor Density CR1 Expression Alzheimer's Systemic Lupus Erythematosus AIDS Malaria Immunostaining Fluorescence Microscopy PBS-BSA Biotinylated Anti-CR1 J3D3 Antibody Streptavidin Phycoerythrin