Published: May 19th, 2023
This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.
The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.
Adipose tissue biology has been attracting ever-increasing attention because of the growing prevalence of obesity and type 2 diabetes globally1. Adipocytes store excess energy in the form of lipid droplets, which are released upon starvation. Moreover, adipose tissue maintains systemic energy homeostasis by serving as an endocrine organ and communicating with other tissues2,3. Intriguingly, both excess adipose tissue (obesity) and adipose loss (lipodystrophy) are linked to insulin resistance and diabetes1. Adipocytes are divided into three types: white, brown, an....
All animal experiments described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tokyo and performed according to the institutional guidelines of the University of Tokyo.
1. Preparation of enzyme solution and medium
This protocol yields fully differentiated, lipid-laden adipocytes 7 days after inducing adipocyte differentiation. The degree of adipocyte differentiation can be evaluated by the oil red o staining of triglycerides and lipids (Figure 1A), or mRNA expression analysis using qPCR-RT of adipocyte genes, such as the master regulator of adipogenesis Pparg and its target Fabp4 (Figure 1B). To induce beige adipocyte differentiation in vitro, a.......
Here, we described a protocol for isolation of the SVF from murine adipose tissue to obtain preadipocytes and perform adipocyte differentiation in vitro. The use of a tissue dissociator for collagenase digestion decreased experimental variation, decreased the risk of contamination, and increased reproducibility. While this procedure is a critical step within the presented protocol, the process is highly automated and optimization is not needed. However, depending on the mouse age and adipose tissue depot, optimi.......
The authors would like to thank Takahito Wada and Saiko Yoshida (The University of Tokyo, Tokyo, Japan) for their experimental assistance. This work was funded by the following grants to Y.H.: research grant from the University of Tokyo Excellent Young Researcher Program; Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists, grant number 19K17976; grant for the Front Runner of Future Diabetes Research (FFDR) from the Japan Foundation for Applied Enzymology, grant number 17F005; grant from the Pharmacological Research Foundation; grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research; grant from ....
|100 mm dish
|12 well plate
|60 mm dish
|Adipose Tissue Dissociation Kit, mouse and rat
|contents: Enzyme D, Enzyme R, Enzyme A and Buffer A
|Cell strainer 70 µm
|Collagen coated dishes, 100 mm
|Collagen coated dishes, 60 mm
|Collagen I Coat Microplate 6 well
|autoclave before use
|Dissecting Scissors, blunt/sharp
|autoclave before use
|Dissecting Scissors, sharp/sharp
|autoclave before use
|DMEM/F-12, GlutaMAX supplement
|Fetal Bovine Serum (FBS)
|gentleMACS C Tubes
|gentleMACSOcto Dissociator with Heaters
|Humulin R Injection U-100
|pBABE-neo largeTcDNA (SV40)
|Platinum-E (Plat-E) Retroviral Packaging Cell Line
|Power Sybr Green Master Mix
|ReverTra Ace qPCR RT Master Mix
|RNeasy Mini Kit (250)
Copyright © 2024 MyJoVE Corporation. All rights reserved