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We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.
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.
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 &....
All animal experimental procedures were performed under approval by the Mayo Clinic Institutional Animal Care and Use Committee.
1. Solution preparation
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.......
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
We acknowledge the Mayo Clinic Microscopy Cell Analysis Core Flow Cytometry Facility for assistance with FACS sorting.
....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 |
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