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

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

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

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

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

Full Text

12,882 Views

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

Please note that some of the translations on this page are AI generated. Click here for the English version.

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.

Fonksiyonel ve fenotipik analizi için antijene reaktif B hücreleri tespit etmek ve ettirecek bir manyetik nano-tanecikleri kullanır basit ama etkili bir yöntem tarif edilmiştir.

Bu basit ama etkili prosedürün genel amacı, fenotipik ve fonksiyonel analizleri için manyetik nanopartiküller kullanarak insan periferik kanından nadir antijen bağlayıcı B hücrelerini izole etmek ve zenginleştirmektir. Bu yöntem, in-vivo antijen karşılaşmasından önce ve sonra nadir antijen bağlayıcı B hücrelerinin fenotipi ve biyolojisi ile ilgili immünoloji alanındaki temel soruların yanıtlanmasına yardımcı olabilir. Bu tekniğin en büyük avantajı, özellikle bir stiren plaka veya büyük bir boncuk kullanıldığında, geometri ile ilgili sorunları ortadan kaldırmasıdır.

Ve oldukça büyük başlangıç hücre popülasyonlarında nadir hücreleri izole etmek için hücreleri akış sitometrisi ile izole ederken akış hızı ile ilgili sorunları ortadan kaldırır. 30 ila 50 mililitre tam kanı heparinize kan alma tüplerine topladıktan hemen sonra, kanı steril oda sıcaklığında magnezyum ve kalsiyum içermeyen PBS ile 1: 1 oranında karıştırın. Daha sonra, kan karışımını, yavaş tutarlı pipet tetikleme basıncı kullanarak steril 50 mililitrelik konik bir tüpte 10 mililitre oda sıcaklığında yoğunluk gradyan çözeltisi üzerine katmanlayın.

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