1. Long-Term Culture of Murine Epidermal Keratinocytes
Reto Caldelari, Maja M. Suter, Dominique Baumann, Eliane Müller 
Journal of Investigative Dermatology 
Volume 114, Issue 5, Pages (May 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Calcium-dependent differentiation of long-term mouse keratinocytes from various passages. (A) Immunofluorescence studies using an anti-pan-keratin antibody (LP34, DAKO) revealed the establishment of a well-organized keratin network anchoring to the plasma membrane within 6 h after elevation of the calcium concentration in the medium to 1.2 mM. The stabilization of adhesion structure components at cell–cell boarders indicated formation of cadherin-mediated intercellular adhesion structures within the same time frame, as assessed by E-cadherin staining (DECMA, a kind gift from R. Kemler, Max Plank Institute, Freiburg, Germany) and Desmoglein 3 (Dsg3) staining (our laboratory; antibodies were raised in rabbits against the extracellular domain of baculovirus-expressed human Dsg3 (Amagai et al. 1994)). To label surface-exposed adhesion molecules, cells were incubated with the respective antibodies prior to fixation and permeabilization. Expression of proteins can be considered as semiquantitative since staining conditions and photographic exposure time were held constant. (B) Involucrin expression in keratinocytes cultured under low calcium or high calcium conditions for 3 d was assessed by western blot analysis. Immunoblots of whole cell lysates were probed with anti-involucrin antibody (SY5, a kind gift from F. Watt, Imperial Cancer Research Fund, London, U.K.). To induce differentiation, calcium concentration was raised to 1.2 mM in defined keratinocyte-SFM (KSFM or purge KSFM) or to 1.8 mM in supplemented Williams' Medium E (WME). Purged cells were depleted from additional growth factors 1 d before calcium elevation (purge KSFM: defined keratinocyte-SFM culture medium without supplementation with EGF or choleratoxin). The Ponceau S-staining of the immunoblot was used to evaluate loading and transfer of the proteins.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 1
Calcium-dependent differentiation of long-term mouse keratinocytes from various passages. (A) Immunofluorescence studies using an anti-pan-keratin antibody (LP34, DAKO) revealed the establishment of a well-organized keratin network anchoring to the plasma membrane within 6 h after elevation of the calcium concentration in the medium to 1.2 mM. The stabilization of adhesion structure components at cell–cell boarders indicated formation of cadherin-mediated intercellular adhesion structures within the same time frame, as assessed by E-cadherin staining (DECMA, a kind gift from R. Kemler, Max Plank Institute, Freiburg, Germany) and Desmoglein 3 (Dsg3) staining (our laboratory; antibodies were raised in rabbits against the extracellular domain of baculovirus-expressed human Dsg3 (Amagai et al. 1994)). To label surface-exposed adhesion molecules, cells were incubated with the respective antibodies prior to fixation and permeabilization. Expression of proteins can be considered as semiquantitative since staining conditions and photographic exposure time were held constant. (B) Involucrin expression in keratinocytes cultured under low calcium or high calcium conditions for 3 d was assessed by western blot analysis. Immunoblots of whole cell lysates were probed with anti-involucrin antibody (SY5, a kind gift from F. Watt, Imperial Cancer Research Fund, London, U.K.). To induce differentiation, calcium concentration was raised to 1.2 mM in defined keratinocyte-SFM (KSFM or purge KSFM) or to 1.8 mM in supplemented Williams' Medium E (WME). Purged cells were depleted from additional growth factors 1 d before calcium elevation (purge KSFM: defined keratinocyte-SFM culture medium without supplementation with EGF or choleratoxin). The Ponceau S-staining of the immunoblot was used to evaluate loading and transfer of the proteins.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions