Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Dong Seong Cho; Jason D. Doles

doi:10.3791/61694

JoVE Journal
Biology

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JoVE Journal Biology

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

마우스 전형 지방 조직에서 지방 전구의 준비

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06:17 min

August 25, 2020

DOI: 10.3791/61694-v

Dong Seong Cho¹, Jason D. Doles¹

¹Department of Biochemistry and Molecular Biology,Mayo Clinic

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Overview

This study presents a protocol for isolating highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting. The method facilitates sensitive downstream analyses, such as single-cell RNA sequencing, validating the efficacy of the isolated cells for further characterization.

Key Study Components

Research Area

Cell biology
Adipose tissue analysis
Single-cell studies

Background

Adipose progenitor cells are crucial for understanding fat biology.
Previous studies lacked effective isolation methods for these cells.
This protocol improves cell viability and purity for subsequent analyses.

Methods Used

Fluorescence activated cell sorting (FACS)
Mouse epididymal adipose tissue as the biological system
Flow cytometry for cell characterization

Main Results

Effective isolation of adipose progenitor cells from male FVB mice.
High viability and purity of the isolated cells were confirmed through flow cytometry.
The method was validated against single-cell RNA sequencing standards.

Conclusions

The study demonstrates a reliable approach to isolate adipose progenitor cells.
This advancement is relevant for future research in cell biology and adipose tissue studies.

Frequently Asked Questions

What are adipose progenitor cells?

Adipose progenitor cells are precursors to adipocytes and play a vital role in the regulation of fat tissue.

Why is the isolation of these cells important?

Isolating these cells allows for detailed investigation into their functions and role in metabolism.

How does FACS improve isolation?

FACS allows for the precise sorting of cells based on specific surface markers, ensuring high purity.

What downstream analyses can be done post-isolation?

Downstream analyses include single-cell RNA sequencing, quantitative real-time PCR, and flow cytometry.

Is the protocol applicable to other animal models?

The protocol was specifically validated in male FVB mice, but it may be adapted for other models.

How long does the entire isolation process take?

The isolation process takes approximately two hours, including incubation and sorting time.

Can this method be used for clinical studies?

While the protocol is designed for mouse models, similar techniques may be adapted for clinical research on human adipose tissue.

우리는 형광 활성화 세포 분류를 사용하여 마우스 전도성 지방 패드에서 매우 실행 가능한 지방 전구 세포를 분리하는 간단한 방법을 제시한다.

이 프로토콜은 민감한 다운스트림 분석을 위해 마우스 부고환 지방 조직에서 고품질 지방 전구 세포를 분리할 수 있습니다. 이 기술은 생존력이 높은 지방 전구 세포를 생성하며 최근 단일 세포 RNA 염기서열 분석 연구에서 검증되었습니다. 수컷 쥐를 안락사시킨 후 부고환 지방 패드를 찾아 뭉툭한 집게로 부드럽게 당겨 신중히 관찰합니다.

가위를 사용하여 고환을 제거하고 50ml 원추형 튜브에 3% BSA가 보충된 HBSS 5ml에 부고환 지방 패드를 15분 동안 배양합니다. 배양 후 150 x g에서 튜브를 섭씨 4도에서 7분 동안 원심분리합니다. 원뿔형 튜브에서 떠 있는 부고환 지방 패드를 제거하고 깨끗한 가위로 잘게 다집니다.

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