A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography.
Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.
Okumura
H
H
Department of Dermatology, Osaka University School of Medicine, Japan.
Matsumto
K
K
Hashimoto
K
K
Yoshikawa
K
K
D002454:Cell Differentiation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011498:Protein Precursors / Q000096:biosynthesis*
D011498:Protein Precursors; C032253:involucrin