کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8418380 | 1545721 | 2012 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Characterizing and quantifying leukocyte populations in human adipose tissue: Impact of enzymatic tissue processing
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کلمات کلیدی
DAPISVCrMFIrelative mean fluorescence intensityAdipose tissue - بافت چربیATM - خودپردازdiamidino-2-phenylindole - دیامیدینو 2-فنیلینولCoefficient of Variation - ضریب تغییرLeukocytes - لکوسیتAdipose tissue macrophage - ماکروفاژ بافت چربیNormalization - نرمالیزاسیون یا عادی سازیEnzymatic digestion - هضم آنزیمی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
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چکیده انگلیسی
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 CD45pos 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 386, Issues 1â2, 14 December 2012, Pages 50-59
Journal: Journal of Immunological Methods - Volume 386, Issues 1â2, 14 December 2012, Pages 50-59
نویسندگان
Derek K. Hagman, Jessica N. Kuzma, Ilona Larson, Karen E. Foster-Schubert, Ling-Yu Kuan, Andrea Cignarella, Elena Geamanu, Karen W. Makar, Jourdan R. Gottlieb, Mario Kratz,