Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Mia J. Smith; Thomas A. Packard; Shannon K. O'Neill; Rochelle M. Hinman; Marynette Rihanek; Peter A. Gottlieb; John C. Cambier

doi:10.3791/55382

Rilevamento e l'arricchimento di Rare antigene-specifiche cellule B per l'analisi del fen...

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Immunology and Infection

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JoVE Journal Immunology and Infection

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Rilevamento e l'arricchimento di Rare antigene-specifiche cellule B per l'analisi del fenotipo e funzione

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09:25 min

February 16, 2017

DOI: 10.3791/55382-v

Mia J. Smith^1,2, Thomas A. Packard¹, Shannon K. O'Neill¹, Rochelle M. Hinman¹, Marynette Rihanek³, Peter A. Gottlieb³, John C. Cambier^1,4

¹Department of Immunology and Microbiology,University of Colorado School of Medicine, ²Department of Microbiology, Immunology, and Pathology,Colorado State University, ³Barbara Davis Center for Childhood Diabetes,University of Colorado School of Medicine, ⁴Department of Biomedical Research,National Jewish Health

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Overview

This article describes a method for isolating and enriching rare antigen-binding B cells from human peripheral blood using magnetic nanoparticles. This technique facilitates their phenotypic and functional analysis, providing insights into the biology of these cells.

Key Study Components

Area of Science

Immunology
Cell Biology
Biotechnology

Background

Antigen-reactive B cells are crucial for understanding immune responses.
Isolating these cells can be challenging due to their rarity.
Traditional methods may introduce geometric and flow rate issues.
This method aims to overcome these challenges using magnetic nanoparticles.

Purpose of Study

To develop a reliable method for enriching antigen-binding B cells.
To analyze the phenotype and function of these cells.
To enhance understanding of B cell biology before and after antigen exposure.

Methods Used

Collection of whole blood into heparinized tubes.
Mixing blood with magnesium and calcium-free PBS.
Layering the mixture onto a density gradient solution.
Utilizing magnetic nanoparticles for cell isolation.

Main Results

The method effectively isolates rare antigen-binding B cells.
It minimizes issues related to geometry and flow rates.
Allows for detailed phenotypic and functional analysis.
Provides a framework for further immunological studies.

Conclusions

This technique is a significant advancement in B cell research.
It opens new avenues for studying immune responses.
Future research can build on this method to explore B cell functions.

Frequently Asked Questions

What are antigen-binding B cells?

Antigen-binding B cells are immune cells that recognize and bind to specific antigens, playing a key role in the immune response.

How does the magnetic nanoparticle method work?

This method uses magnetic nanoparticles to selectively isolate B cells that bind to specific antigens, allowing for their enrichment and analysis.

What are the advantages of this method?

It overcomes challenges related to geometry and flow rates in traditional isolation methods, making it more efficient for rare cell populations.

Can this method be applied to other cell types?

While this method is designed for B cells, similar techniques may be adapted for other immune cell types.

What implications does this research have for immunology?

It enhances our understanding of B cell biology and could lead to new insights into immune responses and therapies.

Un metodo semplice ma efficace che si avvale di nanoparticelle magnetiche per rilevare e arricchire le cellule B antigene-reattiva per l'analisi funzionale e fenotipica è descritto.

L'obiettivo generale di questa procedura semplice ma efficace è isolare e arricchire le cellule B leganti l'antigene raro dal sangue periferico umano utilizzando nanoparticelle magnetiche per la loro analisi fenotipica e funzionale. Questo metodo può aiutare a rispondere a domande chiave nel campo dell'immunologia riguardanti il fenotipo e la biologia delle cellule B leganti l'antigene raro, sia prima che dopo l'incontro con l'antigene in vivo. Il vantaggio principale di questa tecnica è che ovvia ai problemi associati alla geometria, in particolare quando si utilizza una lastra di stirene o un cordone di grandi dimensioni.

E ovvia ai problemi associati alla velocità di flusso quando si isolano le cellule mediante citometria a flusso per isolare cellule rare in popolazioni cellulari iniziali piuttosto grandi. Immediatamente dopo aver raccolto da 30 a 50 millilitri di sangue intero in provette per la raccolta del sangue eparinizzate, mescolare il sangue in un rapporto 1:1 con magnesio sterile a temperatura ambiente e PBS privo di calcio. Quindi, sovrapporre la miscela di sangue su 10 millilitri di soluzione a gradiente di densità a temperatura ambiente in una provetta conica sterile da 50 millilitri utilizzando una pressione di attivazione lenta e costante della pipetta.

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