Flow Cytometric Method for the Detection of Flavonoids in Cell Lines.
Here, we describe an easy-to-use flow cytometric method using diphenylboric acid 2-amino ethyl ester (DPBA) stain for the detection of flavonoids in cells from human/animal origin. Flavonoid bioavailability and bioactivity depend on structure, conjugation and the cell type to which they are presented. We have studied cellular uptake of five flavonoids with different structures and conjugation forms. First, parameters including fixation method, technical and batch variability, and concentration were optimized. Second, uptake of two aglycones-quercetin and hesperetin-and their corresponding glycosides-rutin and hesperidin-in Caco-2 cells was compared. Third, the aglycone quercetin, glycoside rutin, and glucuronide baicalin were added to the Caco-2, HepG2, and CHO-K1 cell lines at 1, 10, and 20 µM concentrations and cellular uptake was measured after 1, 4, and 7 h. We conclude that quercetin was taken up by cells in a dose-dependent way, and that HepG2 cells had the highest uptake factors, followed by CHO-K1 and Caco-2 cells. Confocal microscopy showed cell type-dependent localization of quercetin in the cell membrane and cytoplasm. No uptake of flavonoid glycosides was detected. This flow cytometric method can be used for future research unravelling mechanisms behind flavonoid bioactivity in health and disease at the cellular level.
Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Charlotte
C
Grootaert
Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Department of Industrial Biological Science, Faculty of Bioscience Engineering, Ghent University, Kortrijk, Belgium Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Gerard Bryan
GB
Gonzales
Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Hanne
H
Vissenaekens
Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Tom
T
Van de Wiele
Department of Industrial Biological Science, Faculty of Bioscience Engineering, Ghent University, Kortrijk, Belgium.
Katleen
K
Raes
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Guy
G
Smagghe
Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium john.vancamp@ugent.be.
John
J
Van Camp
D000818:Animals; D001692:Biological Transport / Q000235:genetics*; D016466:CHO Cells; D018938:Caco-2 Cells; D002460:Cell Line / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005419:Flavonoids / Q000302:isolation & purification* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D011794:Quercetin / Q000494:pharmacology
D005419:Flavonoids; D011794:Quercetin
DPBA; cell lines; flavonoid; flow cytometry