TY - JOUR
T1 - Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content
AU - Morgan-Bathke, Maria
AU - Harteneck, Debra
AU - Jaeger, Philippa
AU - Sondergaard, Esben
AU - Karwoski, Ron
AU - Espinosa De Ycaza, Ana
AU - Carranza-Leon, B. Gisella
AU - Faubion, William A.
AU - Oliveira, Andre M.
AU - Jensen, Michael D.
N1 - Funding Information:
We would like to thank the personnel of the Mayo Clinic Clinical Research and Trials Unit, the Mayo Clinic Rochester Pathology Research Cores, and the Flow Cytometry and Cell Sorting Microscopy team of the Cell Analysis Core, as well as Ms Monica Davis for editorial assistance and our volunteers.
Funding Information:
Funding agencies: These studies were supported by National Center for Research Resources Grant 1UL1RR024150 and National Institutes of Health Grants DK45343, DK40484, DK50456, and 5T32 DK007352. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosure: Mr. Karwoski is a salaried employee of Biomedical Imaging Resource, Mayo Clinic, Rochester, Minnesota, which developed the AMCounter program. Potential sales of the AMCounter program could reduce the annual license Dr. Jensen pays to use AMCounter. The other authors declared no conflict of interest. Additional Supporting Information may be found in the online version of this article. Received: 19 December 2016; Accepted: 19 August 2017; Published online 6 October 2017. doi:10.1002/oby.22012
Publisher Copyright:
© 2017 The Obesity Society
PY - 2017/12
Y1 - 2017/12
N2 - Objective: The relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) of ATM markers. Methods: A new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT-PCR. Results: IHC had good intraindividual reproducibility for total (CD68), proinflammatory (CD14), and anti-inflammatory (CD206) ATM. The AMCounter improved interreader agreement and was more time efficient. Flow cytometry had acceptable intraindividual reproducibility for the percentage of CD68+ cells that were CD14+ or CD206+, but not for ATMs per gram of tissue. ATMs per gram of tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and CD206 ATMs per 100 adipocytes. Also of interest were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and adipose flow cytometry measures of CD68+, CD68+/CD14+, and CD68+/CD206+ ATMs per gram of tissue. Conclusions: The AMCounter software helps provide reproducible and efficient measures of IHC ATMs. Flow cytometry, IHC, and RT-PCR measures of adipose inflammation provide somewhat different information.
AB - Objective: The relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) of ATM markers. Methods: A new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT-PCR. Results: IHC had good intraindividual reproducibility for total (CD68), proinflammatory (CD14), and anti-inflammatory (CD206) ATM. The AMCounter improved interreader agreement and was more time efficient. Flow cytometry had acceptable intraindividual reproducibility for the percentage of CD68+ cells that were CD14+ or CD206+, but not for ATMs per gram of tissue. ATMs per gram of tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and CD206 ATMs per 100 adipocytes. Also of interest were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and adipose flow cytometry measures of CD68+, CD68+/CD14+, and CD68+/CD206+ ATMs per gram of tissue. Conclusions: The AMCounter software helps provide reproducible and efficient measures of IHC ATMs. Flow cytometry, IHC, and RT-PCR measures of adipose inflammation provide somewhat different information.
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U2 - 10.1002/oby.22012
DO - 10.1002/oby.22012
M3 - Article
C2 - 28985040
AN - SCOPUS:85035024450
SN - 1930-7381
VL - 25
SP - 2100
EP - 2107
JO - Obesity
JF - Obesity
IS - 12
ER -