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 to 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.

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