Direct evaluation of fluorescence in single renal epithelial cells using a mitochondrial probe (DASPMI)

Abstract

K E Y w ORDS. Kidney, mitochondria, fluorescence, fluorometry, cell culture, loop of Henle, collecting tubule. S U M M A R YThe study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developed in vitro culture system of renal epithelial cells.Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum-free, hormone-supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between C T and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium-range intensity in TAL cell cultures while C T cultures exhibit a two-peak pattern of mitochondrial fluorescence distribution among epithelial cells.

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia

“…Although the majority of CCT cultures showed little reaction for cytochrome oxidase, some cells were seen which stained strongly, suggesting the occurrence of both mitochondrial-poor (principal) cells and mitochondrial-rich (intercalated) cells. This has also been suggested by DASPMI mitochondria1 fluorescent dye uptake studies (Horster et al, 1983a) and transmission electron microscopy (Wilson, unpublished observation) and mirrors the occurrence of these two cell types in CCT in vivo.…”

Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers

“…Previous work has substantiated that the fluorescence signals detected at 366-nm excitation originate from the mitochondrial NADH, whereas the fluorescence signals recorded at 488-nm argon ion laser excitation are largely due to the emission of the oxidized flavin moiety of the mitochondrial flavoprotein α-lipoamide dehydrogenase ( Kunz et al, 1994 ). We show here that the flavoprotein fluorescence obtained in the oxidized state of the myofiber overlaps to a large extent with the fluorescence of the mitochondrial potentiometric dye DASPMI ( Horster et al, 1983 ; Bereiter-Hahn and Voth, 1994 ) and also with MitoTracker™ Green FM ( Hoth et al, 1997 ), which is a constitutive marker of mitochondria. The fluorescence images obtained by confocal microscopy show the typical pattern of mitochondrial organization beneath the sarcolemma (SSM) and along the myofibrils (IMM).…”

“…Hitherto microscopic investigation of mitochondria have been performed mainly using Δψ-dependent fluorescent dyes, like rhodamine 123 ( Chen, 1989 ) or dimethylaminostyryl pyridyl methyl iodide ( Horster et al, 1983 ; Bereiter-Hahn and Voth, 1994 ). There are only a few reports describing the application of fluorescence microscopy for the measurement of the redox state of the mitochondrial NADH at the cellular level ( Eng et al, 1989 ; Piston et al, 1995 ).…”

Fluorescent probes that stain living nerve terminals

“…Moreover, cationic rhodamines affected the function and viability of mammalian myocardial cells in culture (Lampidis et al, 1984) and inhibited oxidative phosphorylation (Gear, 1974;Mai and Allison, 1983). On the other hand, studies of the effects of styryl pyridinium dyes on cultured amphibian myocardial cells (Bereiter-Hahn, 1976), mammalian brown fat (Rafael and Nicholls, 1984), and mammalian renal epithelial cells (Horster et al, 1983) did not reveal any obvious damage. Stained cells continued to grow in culture after dye uptake, and protozoan motility was unaffected by a 24 hr exposure to a styryl pyridinium dye very similar to the reagent we studied in detail (4-Di-2-ASP) (Morozova et al, 1981;Miyakawa et al, 1984).…”