Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Linlin Zhang; Mengjia Liu; Meng Zhang; Junhong Ai; Jiao Tian; Ran Wang; Zhengde Xie

doi:10.3791/64212

Separazione delle sottopopolazioni di cellule immunitarie nei campioni di sangue periferico da ba...

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Immunology and Infection

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JoVE Journal Immunology and Infection

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Separazione delle sottopopolazioni di cellule immunitarie nei campioni di sangue periferico da bambini con mononucleosi infettiva

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08:44 min

September 7, 2022

DOI: 10.3791/64212-v

Linlin Zhang^1,2, Mengjia Liu^1,2, Meng Zhang^1,2, Junhong Ai^1,2, Jiao Tian^1,2, Ran Wang^1,2, Zhengde Xie^1,2

¹Beijing Key Laboratory of Pediatric Respiratory Infectious Diseases, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital,Capital Medical University, National Center for Children's Health, ²Research Unit of Critical Infection in Children, 2019RU016,Chinese Academy of Medical Sciences

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Overview

This article describes a method that combines immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze specific immune cell subpopulations from peripheral blood mononuclear cells. The method allows for the purification and analysis of magnetic and fluorescently labeled cells.

Key Study Components

Area of Science

Immunology
Cell Biology
Flow Cytometry

Background

Peripheral blood mononuclear cells (PBMCs) are crucial for studying immune responses.
Understanding immune cell subpopulations can provide insights into various diseases.
EBV infection mechanisms can be better characterized through this method.
Isolation of specific cell types enhances the ability to study their functions and interactions.

Purpose of Study

To isolate and analyze defined immune cell subpopulations.
To expand understanding of immune responses in diseases.
To demonstrate the procedure for isolating PBMCs effectively.

Methods Used

Isolation of PBMCs using centrifugation.
Use of CD14 microbeads for monocyte isolation.
Fluorescence-activated cell sorting for analysis.
Washing and resuspending cells in a prepared buffer.

Main Results

Successful isolation of specific immune cell types.
Enhanced ability to analyze immune responses in individuals.
Demonstration of the method's effectiveness in a laboratory setting.
Potential applications in studying EBV infection mechanisms.

Conclusions

The method provides a reliable approach for isolating immune cells.
It can significantly contribute to research on immune responses.
Future studies can leverage this method to explore various diseases.

Frequently Asked Questions

What are PBMCs?

PBMCs, or peripheral blood mononuclear cells, are blood cells that have a round nucleus and include lymphocytes and monocytes.

How does fluorescence-activated cell sorting work?

Fluorescence-activated cell sorting (FACS) uses fluorescent markers to identify and sort specific cell types based on their characteristics.

What is the significance of isolating immune cell subpopulations?

Isolating immune cell subpopulations allows researchers to study their specific functions and roles in immune responses and diseases.

Can this method be applied to other types of cells?

Yes, the method can be adapted to isolate and analyze other cell types beyond immune cells.

What diseases can benefit from this research?

This research can benefit studies on infectious diseases, autoimmune disorders, and cancer.

Descriviamo un metodo che combina sfere immunomagnetiche e selezione cellulare attivata dalla fluorescenza per isolare e analizzare sottopopolazioni definite di cellule mononucleate del sangue periferico (monociti, cellule T CD4 +, cellule T CD8 ⁺, cellule B e cellule natural killer). Utilizzando questo metodo, le cellule magnetiche e fluorescenti possono essere purificate e analizzate.

Questo metodo consentirà la caratterizzazione di diverse cellule immunitarie negli stessi stati di malattia di individui con IM, che amplierà la nostra comprensione del meccanismo dell'infezione da EBV. A dimostrare la procedura sarà Linlin Zhang, un medico del nostro laboratorio. Per iniziare, prendere i PBMC isolati in precedenza in una provetta da microcentrifuga da 1,5 millilitri e ruotarli a 300 G per 10 minuti a temperatura ambiente.

Scartare il surnatante e risospendere le cellule con 80 microlitri del tampone preparato. Quindi, aggiungere 20 microlitri di microsfere CD 14 alla sospensione cellulare, mescolata bene mediante pipettaggio, e incubare il tubo per 15 minuti sul ghiaccio. Quindi lavare le PBMC utilizzando un millilitro di tampone e centrifugare a 300 G per 10 minuti a temperatura ambiente.

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