Analysis and isolation of adipocytes by flow cytometry.
Analysis and isolation of adipocytes via flow cytometry is particularly useful to study their biology. However, the adoption of this technology has often been hampered by the presence of stromal/vascular cells in adipocyte fractions prepared from collagenase-digested adipose tissue. Here, we describe a multistep staining method and gating strategy that effectively excludes stromal contaminants. Initially, we set a gate optimized to the size and internal complexity of adipocytes. Exclusion of cell aggregates is then performed based on fluorescence of a nuclear stain followed by positive selection to collect only those cell events containing lipid droplets. Lastly, negative selection of cells expressing stromal or vascular lineage markers removes any remaining stromal contaminants. These procedures are applicable to simple analysis of adipocytes and their subcellular constituents by flow cytometry as well as isolation of adipocytes by flow sorting.
Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Susan M
SM
Majka
Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Heidi L
HL
Miller
Cancer Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Karen M
KM
Helm
Cancer Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Alistaire S
AS
Acosta
Cancer Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Christine R
CR
Childs
Amnis Corporation, Seattle, Washington, USA.
Raymond
R
Kong
Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address: dwight.klemm@ucdenver.edu.
Dwight J
DJ
Klemm
D017667:Adipocytes / Q000166:cytology*; D000273:Adipose Tissue / Q000166:cytology; D015415:Biomarkers; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics*; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans
D015415:Biomarkers
Adipocyte; Adipose tissue; Flow cytometry; Flow sorting; Obesity