Long-term staining of live Merkel cells with FM dyes.
Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. To find dyes that remain in the cells for a long period of time, we tested many fluorescence dyes and found that FM dyes (such as FM1-43) are useful markers for live Merkel cells. In the rat footpad skin, FM1-43 was shown to stain 95% of live Merkel cells that were already stained with quinacrine. FM4-64 stained 98% of quinacrine-stained Merkel cells. Merkel cells in sinus hair follicles were also stained with FM dyes. The fluorescence intensity of FM dyes was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM-dye-stained cells. To test how long FM dyes remain in live cells, FM-dye-stained Merkel cells in hair follicles were embedded in collagen gel and were cultured in a serum-free medium. FM-dye-stained cells were easily identified even after 7 days of culture. During the culture, Merkel cells changed their shape, moved in the preparation and tended to aggregate on the surface. We conclude that FM dyes are powerful tools for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate FM dyes suggests that vesicles in the cells are likely to have mechanisms of recycling in a manner similar to those in neurons and secretory cells.
Fukuda
Jun
J
Laboratory of Molecular and Cellular Physiology, Department of Physiology, National Defense Medical College of Japan, 3-2 Namiki, Tokorozawa, 359-8513, Saitama, Japan. jfukuda@cc.ndmc.ac.jp
Ishimine
Hisako
H
Masaki
Yoshinori
Y
D000818:Animals; D000831:Animals, Newborn; D016477:Artifacts; D048429:Cell Size / Q000502:physiology; D002478:Cells, Cultured; D046508:Culture Techniques / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D018859:Hair Follicle / Q000166:cytology* / Q000294:innervation*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D053554:Keratin-20; D007633:Keratins; D018862:Merkel Cells / Q000166:cytology* / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods / Q000639:trends; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D011796:Quinacrine; D051381:Rats; D015203:Reproducibility of Results; D012867:Skin / Q000166:cytology* / Q000294:innervation*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
C113793:FM 1 84; C092350:FM 4-64; C073804:FM1 43; D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins; D053554:Keratin-20; C489973:Krt20 protein, rat; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D007633:Keratins; D011796:Quinacrine