Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Alberto Y&#225;&#241;ez; Helen S. Goodridge

doi:10.3791/58061

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

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07:21 min

July 29, 2018

DOI: 10.3791/58061-v

Alberto Yáñez^1,2, Helen S. Goodridge^1,2

¹Board of Governors Regenerative Medicine Institute,Cedars-Sinai Medical Center, ²Research Division of Immunology,Cedars-Sinai Medical Center

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Overview

This article demonstrates a method for identifying and isolating six subsets of myeloid progenitors from murine bone marrow using magnetic and fluorescence sorting techniques. The protocol is applicable for various assays, including in vitro culture and RNA/protein analyses.

Key Study Components

Area of Science

Hematology
Immunology
Cell Biology

Background

Myeloid cells play a crucial role in the immune response.
Understanding myeloid progenitor subsets is essential for studying their functions.
Previous methods lacked precision in identifying these subsets.
This study introduces a refined technique for better identification.

Purpose of Study

To provide a reliable method for isolating myeloid progenitor subsets.
To enhance the understanding of myeloid cell production and function.
To facilitate further research in hematology and immunology.

Methods Used

Magnetic-activated cell sorting (MACS) for cell enrichment.
Fluorescence-activated cell sorting (FACS) for precise identification.
In vitro culture assays using methylcellulose or liquid cultures.
RNA and protein analyses for functional studies.

Main Results

Successful isolation of six distinct myeloid progenitor subsets.
Improved accuracy in identifying progenitor cells compared to previous methods.
Protocol applicable for various downstream applications.
Potential to advance research in myeloid cell biology.

Conclusions

The method provides a significant advancement in myeloid progenitor research.
It opens new avenues for studying myeloid cell functions.
Researchers can utilize this protocol for diverse experimental applications.

Frequently Asked Questions

What are myeloid progenitors?

Myeloid progenitors are precursor cells that give rise to various types of myeloid cells, including neutrophils and monocytes.

How does MACS work?

MACS uses magnetic beads coated with antibodies to isolate specific cell types from a mixture based on their surface markers.

What is the significance of isolating myeloid progenitor subsets?

Isolating these subsets allows researchers to study their specific roles and functions in the immune system.

Can this method be applied to human samples?

While this protocol is designed for murine models, similar techniques can be adapted for human samples with appropriate modifications.

What downstream applications can this protocol support?

The protocol can support in vitro culture assays, RNA/protein analyses, and in vivo adoptive transfer experiments.

Is prior experience required to use this method?

Some experience with cell sorting techniques and basic laboratory skills is recommended for optimal results.

識別し、並べ替え (MAC および FACS) 蛍光と磁気の組み合わせを使ってマウスの骨髄から骨髄前駆細胞の 6 のサブセットを分離する方法を示します。このプロトコルは、体外培養の試金 (セルロースまたは液体文化)、養子の転送実験生体内RNA ・タンパク質解析に使用できます。

この方法は、好中球、単球、樹状細胞などの骨髄系細胞の産生と機能を研究する血液学者や免疫学者に役立つ可能性があります。この手法の主な利点は、以前の戦略よりも骨髄系前駆細胞サブセットをより正確に同定できることです。磁気活性化細胞選別(MACS)により前駆細胞の骨髄細胞を濃縮するには、細胞を遠心分離によりペレット化し、ペレットを7つの骨髄細胞の10倍あたり40マイクロリットルのMACS染色緩衝液に再懸濁します。

分化した系統陽性細胞を枯渇させるには、7つの細胞に10×10あたり10マイクロリットルのビオチン抗体カクテルを加え、ピペッティングで摂氏4度で10分間インキュベーションして混合します。インキュベーション終了時に、7つの細胞に1×10あたり30マイクロリットルの染色緩衝液を混合しながら加え、続いて7つの細胞に1×10あたり20マイクロリットルの抗ビオチンマイクロビーズを添加します。摂氏4度で15分後、7つの細胞に1×10あたり1ミリリットルの染色緩衝液で細胞を洗浄し、ペレットを500マイクロリットルの新鮮な染色緩衝液に再懸濁します。

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