Fluorescent activated cell sorting to isolate canine microvascular endothelial cells from adipose tissue

Renee K. Allen, Tonya T. Reddick, Diana M. Popp, Robert Donnell, Michael Freeman, Scott Stevens, Mitchell Goldman

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Adipose tissue offers an abundant source for isolation of microvascular endothelial cells (MVECs). Several cell types result from the enzymatic digestion of adipose tissue, including MVECs, mesothelial cells and fibroblasts. Pure populations of MVECs must be isolated from the mixed cultures or fibroblasts overgrow the population. Canine subcutaneous or mesenteric fat is obtained during elective surgical procedures and digested with collagenase. Microvascular endothelial cells are separated from capillary fragments by Percoll gradient centrifugation and plated in culture. At confluence, microscopic identification of most cultured cells indicate the typical cobblestone morphology of ECs, while other spindle shaped cells resemble fibroblasts. Microvascular endothelial cells are identified by their uptake of acetylated-low density lipoprotein (Ac-LDL). Mesothelial cells, which closely resemble MVECs in morphology, and fibroblasts are removed from cultures of MVECs using a fluorescent activated cell sorter (FACS). Acetylated-low density lipoprotein tagged with a fluorescent probe, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), is incubated with the mixed cultures and the cells are sorted using a FACS. The pure populations of MVECs that result are tested immunocytochemically and are identified by their positive staining with antibodies against factor VIII related antigen. Mesothelial cells are identified by positive staining for cytokeratin 18 antibody. Contaminating fibroblasts show negative staining for smooth muscle alpha actin, cytokeratin 18 and factor VIII related antigen antibodies. This study examines the efficiency of the fluorescent activated cell sorter to obtain pure populations of MVECs harvested from adipose tissue.

Original languageEnglish (US)
Pages (from-to)285-294
Number of pages10
JournalMethods in Cell Science
Volume19
Issue number4
DOIs
StatePublished - Dec 1 1998

Fingerprint

Endothelial cells
Sorting
Canidae
Adipose Tissue
Endothelial Cells
Cells
Tissue
Fibroblasts
Antibodies
Keratin-18
Lipoproteins
von Willebrand Factor
Antigens
Cell culture
Population
Elective Surgical Procedures
Staining and Labeling
Negative Staining
Centrifugation
Collagenases

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Fluorescent activated cell sorting to isolate canine microvascular endothelial cells from adipose tissue. / Allen, Renee K.; Reddick, Tonya T.; Popp, Diana M.; Donnell, Robert; Freeman, Michael; Stevens, Scott; Goldman, Mitchell.

In: Methods in Cell Science, Vol. 19, No. 4, 01.12.1998, p. 285-294.

Research output: Contribution to journalArticle

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