Flow Cytometry Analyses of Adipose Tissue Macrophages

Abstract

Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory.

Insulin resistance causes inflammation in adipose tissue

“…SVCs were isolated and stained with antibody as previously described (64). In brief, fat pads were excised and minced in HBSS ++ supplemented with 0.5% BSA and digested with 1 mg/ml type II collagenase (SigmaAldrich) at 37°C for 40 minutes with vigorous shaking.…”

TUSC2 Immunogene Therapy Synergizes with Anti–PD-1 through Enhanced Proliferation and Infiltration of Natural Killer Cells in Syngeneic Kras-Mutant Mouse Lung Cancer Models

“…The cells were stained according to standard protocols for flow cytometry. Single-cell suspensions from the spleen and tumor-nodule–bearing lungs were prepared as described elsewhere (23) and stained for flow cytometry analysis. Multi-color panels were developed and optimized for use with a Gallios Flow Cytometer Research System (Beckman Coulter, Brea, CA).…”

“…(F) NK, (G) Treg, (H) MDSC, and (I) PD-L1 + and PD-L2 + leukocyte infiltration determined by flow cytometry of single cells prepared from metastasized lungs. Data were normalized based on per gram of tumor tissue (23). PD+CT indicate anti–PD-1+anti–CTLA-4 treatment.…”

An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4+ T Cells Controls Obesity-Induced Inflammation

“…SVCs from adipose tissues were isolated as previously described (Cho et al, 2014). Cells were incubated in Fc Block for 10 min on ice and stained with indicated antibodies for 30 min at 4°C.…”