Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
Peer-reviewed

Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro

鼠脂肪组织源间充质基质细胞(mASC)的分离和体外增殖分析

Per Anderson Per Anderson
AC Ana Belén Carrillo-Gálvez
FM Francisco Martín

发布: 2015年11月05日第5卷第21期 DOI: 10.21769/BioProtoc.1642 浏览次数: 10962

评审: Agnieszka PastulaAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Jan 2015

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

评估小鼠脾脏中造血干细胞和祖细胞的存在

评估小鼠脾脏中造血干细胞和祖细胞的存在

Isabelle J. Marié [...] David E. Levy

2022年06月05日 2772 阅读

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

Chloe A. L. Thompson-Peach [...] Daniel Thomas

2023年01月20日 1328 阅读

用于单细胞RNA测序的人骨髓非造血细胞的分离

用于单细胞RNA测序的人骨髓非造血细胞的分离

Hongzhe Li [...] Stefan Scheding

2024年06月20日 1361 阅读

Abstract

Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.

Materials and Reagents

  1. Scalpel blades No. 24 (VWR International, catalog number: 233-5487 )
  2. Cell strainers (100 μm and 70 μm) (BD Biosciences, catalog numbers: 352360 and 352350 )
    Note: Currently, it is “Corning , catalog numbers: 352360 and 352350”.
  3. Black 96-well plate (Greiner Bio-One GmbH, catalog number: 655076 )
  4. 16-well E-plate (E-plate 16) (ACEA BIO, catalog number: 06465382001 )
  5. Tissue culture treated Falcon 96-well plates (BD Biosciences, catalog number: 353072 )
    Note: Currently, it is “Corning, catalog number: 353072”.
  6. NuncTM Cell Culture Treated EasYFlasksTM (Thermo Fisher Scientific, catalog number: 156499 )
  7. BALB/c (or any strain of choice) 8-12 weeks old male or female mice (Charles River Laboratories)
  8. Hank’s balanced salt solution (HBSS) w/Calcium w/Magnesium (Biowest, catalog number: L0612-500 )
  9. Collagenase Type I (Sigma-Aldrich, catalog number: C0130-100MG )
  10. MesenCultTM Proliferation Kit with MesenPureTM (Mouse) (STEMCELL Technologies, catalog number: 05512 )
  11. Penicillin-Streptomycin 10,000 U/ml (Life Technologies, Gibco, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 15140-122”.
  12. Stable Glutamine 200 mM (Biowest, catalog number: X0551-100 )
  13. DPBS w/o Calcium w/o Magnesium (Biowest, catalog number: L0615-500 )
  14. TrypLETM Express Enzyme (1x) (Life Technologies, catalog number: 12604021 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 12604021”.
  15. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: 10309433 )
  16. Trypan blue (Sigma-Aldrich, catalog number: T8154-20 ml )
  17. CellTiter-Blue reagent (Promega Corporation, catalog number: G8081 )
  18. Collagenase Type I (see Recipes)
  19. Complete MesenCult medium (see Recipes)

Equipment

  1. Sterile stainless steel scissors and forceps
  2. Neubauer cell counting chamber (VWR International, catalog number: 631-1111 )
  3. Cell culture incubator (THERMO, model: HEPA class 100 )
  4. Phase contrast microscope (OLYMPUS, model: CKX41 )
  5. Centrifuge for cells (Eppendorf, model: 5810R )
  6. xCELLigence RTCA station and control unit (lap top) with the RTCA Software (ACEA BIO/ROCHE)
  7. Fluorometro [Glomax multidetection system (Promega Corporation) or equivalent]

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Anderson, P., Carrillo-Gálvez, A. B. and Martín, F. (2015). Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro. Bio-protocol 5(21): e1642. DOI: 10.21769/BioProtoc.1642.

ris ris Download Citation in RIS Format

分类

干细胞 > 成体干细胞 > 造血干细胞

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件