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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Abstract

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies – including single-cell RNA-sequencing and single-cell proteomics – is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

Introduction

Adipose tissue plays a key role in energy metabolism. Excess energy is stored in the form of lipids, and adipose tissue is capable of significant expansion or retraction depending on nutritional status and energetic demand. Expansion of adipose tissue can result from an increase in adipocyte size (hypertrophy) and/or from an increase in adipocyte number (hyperplasia); the latter process tightly regulated by proliferation and differentiation of adipose progenitor cells1,2. During obesity, adipose tissue excessively expands, and tissue dysfunction – including hypoxia, inflammation, and insulin resistance &....

Protocol

All animal experimental procedures were performed under approval by the Mayo Clinic Institutional Animal Care and Use Committee.

1. Solution preparation

  1. Prepare collagenase 2% (w/v) solution by dissolving collagenase II 2 g in 100 mL Hanks' balanced salt solution (HBSS). Aliquot 200 µL each for each use.
  2. Prepare neutralization medium by mixing 84 mL F-12 medium, 15 mL horse serum, and 1 mL penicillin/streptomycin.
  3. Prepare flow cytometr.......

Representative Results

Four-month-old male FVB mice were used in this experiment. After exclusion of debris and doublets using FSC/SSC plots, viable cells (DAPI- population) were gated, followed by the selection of APC+/FITC- population (Figure 1). DAPI, APC, and FITC gates were drawn based on the unstained control. Gating strategies are shown in Figure 1.

After 1 h of sorting, the quality of isolation was quantitatively eva.......

Discussion

Single cell RNA sequencing (scRNA-seq) is rapidly gaining traction as a powerful tool to simultaneously study diverse cell populations at the single cell level. Due to high costs associated with sample preparation and high throughput sequencing, it is imperative to optimize cellular inputs (high viability and purity) to increase the likelihood of experimental success. Some cell preparation protocols rely on removal of dead cells and debris using low-spin washes and column-based separation without FACS sorting

Acknowledgements

We acknowledge the Mayo Clinic Microscopy Cell Analysis Core Flow Cytometry Facility for assistance with FACS sorting.

....

Materials

NameCompanyCatalog NumberComments
1.7 mL microcentrifuge tubeVWR87003-294
13 mL culture tubeThermo Fisher Scientific50-809-216
15 mL conical tubeGreiner Bio-one188 271
5 mL test tube with cell strainer snap capThermo Fisher Scientific08-771-23
50 mL conical tubeGreiner Bio-one227 261
70 µm cell strainerThermo Fisher Scientific22-363-548
Anti-CD31-FITC antibodyMiltenyi Biotec130-102-519
Anti-CD45-FITC antibodyMiltenyi Biotec130-102-491
Anti-Sca1-APC antibodyMiltenyi Biotec130-102-833
Anti-Ter119-FITC antibodyMiltenyi Biotec130-112-908
BSAGold BiotechnologyA-420-500
Collagenase type IIThermo Fisher Scientific17101-015
DAPIThermo FisherD1306
Dulbecco's phosphate-buffered saline (DPBS)Thermo Fisher Scientific14190-144
F-12 mediumThermo Fisher Scientific11765-054
FcR blocking reagentMiltenyi Biotec130-092-575
Hanks' balanced salt solution (HBSS)Thermo Fisher Scientific14025-092
Horse serumThermo Fisher Scientific16050-122
Penicillin-streptomycinThermo Fisher Scientific15140-122
Propidium iodide solutionMiltenyi Biotec130-093-233

References

  1. Krotkiewski, M., Bjorntorp, P., Sjostrom, L., Smith, U. Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution. The Journal of Clinical Investigation. 72, 1150-1162 (1983).
  2. Salans, L. B., Horton, E. S., Sims, E. A.

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Adipose Progenitor CellsMouse Epididymal Adipose TissuesAdipose Tissue IsolationCell IsolationCell SeparationFlow CytometrySca1Ter119CD31CD45

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