Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

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 cells^1,2. 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 cytometry buffer by dissolving 500 mg of bovine serum albumin (BSA) and 400 µL of 0.5 M EDTA in 100 mL of Dulbecco's phosphate-buffered saline (DPBS)....

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

Materials

Name	Company	Catalog Number	Comments
1.7 mL microcentrifuge tube	VWR	87003-294
13 mL culture tube	Thermo Fisher Scientific	50-809-216
15 mL conical tube	Greiner Bio-one	188 271
5 mL test tube with cell strainer snap cap	Thermo Fisher Scientific	08-771-23
50 mL conical tube	Greiner Bio-one	227 261
70 µm cell strainer	Thermo Fisher Scientific	22-363-548
Anti-CD31-FITC antibody	Miltenyi Biotec	130-102-519
Anti-CD45-FITC antibody	Miltenyi Biotec	130-102-491
Anti-Sca1-APC antibody	Miltenyi Biotec	130-102-833
Anti-Ter119-FITC antibody	Miltenyi Biotec	130-112-908
BSA	Gold Biotechnology	A-420-500
Collagenase type II	Thermo Fisher Scientific	17101-015
DAPI	Thermo Fisher	D1306
Dulbecco's phosphate-buffered saline (DPBS)	Thermo Fisher Scientific	14190-144
F-12 medium	Thermo Fisher Scientific	11765-054
FcR blocking reagent	Miltenyi Biotec	130-092-575
Hanks' balanced salt solution (HBSS)	Thermo Fisher Scientific	14025-092
Horse serum	Thermo Fisher Scientific	16050-122
Penicillin-streptomycin	Thermo Fisher Scientific	15140-122
Propidium iodide solution	Miltenyi Biotec	130-093-233