Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 75, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/50707
Keywords
Immunology; Issue 75; Cellular Biology; Molecular Biology; Biophysics; Physiology; Anatomy; Biomedical Engineering; Surgery; Metabolic Diseases; Diabetes Mellitus; diabetes; Endocrine System Diseases; adipose tissue; AT; stromal vascular fraction; macrophage; lymphocyte; T cells; adipocyte; inflammation; obesity; cell; isolation; FACS; flow cytometry; mice; animal model
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Funding
- NIH Ruth L. Kirschstein NRSA [F32 DK091040]
- American Diabetes Association [7-10-MI-05]
- American Heart Association Established Investigator Award [12EIA8270000]
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The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.
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