Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin.
Griffonia simplicifolia agglutinin (GSA) is a valuable histochemical tool in the identification of endothelium. In this study GSA labeled with fluorescein isothiocyanate (GSA-FITC) was used to purify cultures of murine cerebral microvascular endothelium. Cultures were stained with GSA-FITC, then sorted using a fluorescence-activated cell sorter (FACS). GSA-positive endothelial cells were collected, re-cultured, and subsequently re-analyzed by FACS using GSA-FITC. Cultures that initially contained 80 +/- 3 to 89 +/- 3% (X +/- SE) GSA-positive cells were purified to 98 +/- 1% positivity. Immunohistochemistry with an anti-muscle-action antibody confirmed that FACS sorting of GSA-FITC-stained cells effectively removed contaminating smooth muscle cells from endothelial cell cultures. Viability, proliferation, and prostaglandin production of the cells was unaltered by lectin staining and FACS sorting. Thus, GSA-FITC can be used in conjunction with flow cytometry to enhance the purity of murine endothelial cell cultures without adversely affecting cell viability, growth, or metabolism.
Sahagun
G
G
Department of Pathology, University of Iowa, Iowa City 52242.
Moore
S A
SA
Fabry
Z
Z
Schelper
R L
RL
Hart
M N
MN
D000199:Actins / Q000378:metabolism; D000818:Animals; D002455:Cell Division; D002469:Cell Separation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D006651:Histocytochemistry; D037102:Lectins; D051379:Mice; D037121:Plant Lectins; D011453:Prostaglandins / Q000378:metabolism
D000199:Actins; D005452:Fluoresceins; C045475:Griffonia simplicifolia lectins; D037102:Lectins; D037121:Plant Lectins; D011453:Prostaglandins