Characterizing and quantifying leukocyte populations in human adipose tissue: Impact of enzymatic tissue processing

被引:25
作者
Hagman, Derek K. [2 ]
Kuzma, Jessica N.
Larson, Ilona
Foster-Schubert, Karen E. [3 ]
Kuan, Ling-Yu
Cignarella, Andrea [4 ]
Geamanu, Elena
Makar, Karen W.
Gottlieb, Jourdan R. [5 ]
Kratz, Mario [1 ,2 ,3 ]
机构
[1] Fred Hutchinson Canc Res Ctr, Canc Prevent Program, Div Publ Hlth Sci, Seattle, WA 98109 USA
[2] Univ Washington, Sch Publ Hlth, Dept Epidemiol, Seattle, WA 98195 USA
[3] Univ Washington, Dept Med, Div Metab Endocrinol & Nutr, Seattle, WA 98195 USA
[4] Univ Padua, Dept Pharmaceut & Pharmacol Sci, Padua, Italy
[5] Seattle Surg Ctr, Seattle, WA USA
关键词
Adipose tissue; Leukocytes; Enzymatic digestion; Normalization; DIET-INDUCED OBESITY; INDUCED INSULIN-RESISTANCE; C-REACTIVE PROTEIN; T-CELLS; MATURE ADIPOCYTES; STEM-CELLS; TNF-ALPHA; MACROPHAGES; INFLAMMATION; EXPRESSION;
D O I
10.1016/j.jim.2012.08.018
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Adipose tissue inflammation is a major mechanistic link between obesity and chronic disease. To isolate and characterize specific leukocyte populations, e.g. by flow cytometry, tissue needs to be processed to digest the extracellular matrix. We have systematically compared the impact of different commonly used collagenase preparations, digestion times, and normalization strategies on the reproducibility of flow cytometric phenotyping of adipose tissue leukocyte populations. Subcutaneous adipose tissue was obtained from 11 anonymous donors undergoing elective procedures at a plastic surgery clinic in Seattle, WA. We found that collagenase alone consistently produced better cell yields (p = 0.007) than when combined with additional proteases such as the commercially available liberases. Moreover, liberase significantly degraded the cell surface expression of CD4 (p<0.001) on T cells and to a lesser extent CD16 (p = 0.058) on neutrophils. Extension of the digestion interval from 30 to 120 min did not significantly impact cell viability (p = 0.319) or yield (p = 0.247). Normalization by either 'live-gate' or percentage of CD45(pos) leukocytes exhibited the lowest coefficient of variation for tissue digests between 60 and 75 min, compared to normalization per gram of tissue, which consistently exhibited the greatest variability. Our data suggest that digestion of adipose tissue using pure collagenase for 60-75 min provides the best cell yield and viability, with minimal degradation of cell surface markers used to identify immune cell subpopulations, and best reproducibility independent of the normalization strategy. (C) 2012 Elsevier B.V. All rights reserved.
引用
收藏
页码:50 / 59
页数:10
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