Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Muthulekha Swamydas; Michail S. Lionakis

doi:10.3791/50586

İzolasyon, Arıtma ve İşlevsel Araştırmalar ve Kabul edilmiş Transfer deneyler için Fare Kemik İli...

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Immunology and Infection

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JoVE Journal Immunology and Infection

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

İzolasyon, Arıtma ve İşlevsel Araştırmalar ve Kabul edilmiş Transfer deneyler için Fare Kemik İliği Nötrofiller Etiketlenmesi

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07:28 min

July 10, 2013

DOI: 10.3791/50586-v

Muthulekha Swamydas¹, Michail S. Lionakis¹

¹Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases,National Institute of Allergy and Infectious Diseases, NIH

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Overview

This article describes a protocol for isolating and labeling neutrophils from mouse bone marrow. The method involves density gradient centrifugation and the use of CellTracker dyes, providing a reproducible and economical approach for obtaining neutrophils for various studies.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Immunology

Background

Neutrophils are crucial for immune response and inflammation.
Isolation of neutrophils is essential for functional studies.
Bone marrow is a primary source for neutrophil extraction.
Labeling neutrophils aids in tracking their behavior in vivo.

Purpose of Study

To isolate neutrophils from mouse bone marrow.
To label neutrophils for tracking in adoptive transfer experiments.
To facilitate downstream functional studies of neutrophils.

Methods Used

Collection of bone marrow from murine femurs and tibia.
Osmotic lysis of red blood cells using sodium chloride solutions.
Isolation of neutrophils via density gradient centrifugation.
Labeling of neutrophils with CellTracker dyes.

Main Results

Successful isolation of neutrophils from mouse bone marrow.
Effective labeling of neutrophils for tracking purposes.
Flow cytometry can quantify adoptively transferred neutrophils.
Method demonstrates reproducibility and efficiency.

Conclusions

The protocol provides a reliable method for neutrophil isolation.
Labeling enhances the ability to study neutrophil dynamics.
This approach can be applied to various functional studies.

Frequently Asked Questions

What are the main applications of isolated neutrophils?

Isolated neutrophils can be used for functional studies, adoptive transfer experiments, and tracking their behavior in various tissues.

How are red blood cells removed during the isolation process?

Red blood cells are osmotically lysed using sequential sodium chloride solutions before neutrophil isolation.

What is the significance of using CellTracker dyes?

CellTracker dyes allow for the labeling of neutrophils, enabling researchers to track their movement and behavior in vivo.

Can this method be used for neutrophils from other species?

The protocol is optimized for mouse bone marrow neutrophils, but similar methods may be adapted for other species.

What techniques can be used to analyze the isolated neutrophils?

Flow cytometry is commonly used to analyze the number and characteristics of isolated and labeled neutrophils.

Bir yoğunluk dereceli santrifüj yoluyla ve CellTracker boyalar kullanılarak nötrofil etiketlemesi için fare kemik iliğinden nötrofillerin izolasyonu ve saflaştırılması için bir protokol açıklamaktadır. Bu aşağı fonksiyonel çalışmalar ya da evlatlık transferi ve izleme deneyler için nötrofil çok sayıda elde etmek için basit, hızlı, tekrarlanabilir ve ekonomik yöntem temsil eder.

Bu prosedürün genel amacı, aşağı akış fonksiyonel çalışmaları ve evlat edinen transfer deneyleri için fare kemik iliğinden nötrofilleri izole etmek ve etiketlemektir. Bu, önce murin, femur ve tibiadan kemik iliği hücrelerinin toplanmasıyla gerçekleştirilir. İkinci aşamada, kırmızı kan hücreleri sırasıyla% 0.2 ve% 1.6 sodyum klorür çözeltileri içinde sırayla süspanse edilerek ozmotik olarak bağlanır.

Daha sonra, nötrofiller yoğunluk gradyan santrifüjlemesi ile izole edilir. Son adımda, nötrofiller hücre izleyici boyalarla etiketlenir. Sonuç olarak, akış sitometrisi, çeşitli fare dokularına alınan evlat edinici olarak aktarılan nötrofillerin sayısını belirlemek için kullanılabilir.

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