PMID: 8967793Apr 1, 1996Paper

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

Archives of Dermatological Research
C P GladeP C Van de Kerkhof

Abstract

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured ...Continue Reading

References

Jan 1, 1990·Archives of Dermatological Research·S de MareP C van de Kerkhof
Jun 1, 1991·Burns : Journal of the International Society for Burn Injuries·F BoucherR Leloup
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Mar 1, 1988·Clinical and Experimental Dermatology·A ChangP C van de Kerkhof
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Citations

Apr 6, 2004·Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology·Jackie HarneyMichele I Saunders
Oct 10, 1998·The British Journal of Dermatology·J M MommersP C Van De Kerkhof
Mar 24, 2000·The British Journal of Dermatology·J M MommersP C van de Kerkhof
Feb 24, 2004·Biological Chemistry·Maria C RamosPeter Brenneisen
Aug 14, 2003·Biochemical and Biophysical Research Communications·Holger SteinbrennerPeter Brenneisen
May 17, 2007·Brazilian Journal of Biology = Revista Brasleira De Biologia·A GragnaniL M Ferreira
Sep 5, 2002·Experimental and Toxicologic Pathology : Official Journal of the Gesellschaft Für Toxikologische Pathologie·Danielle Roman, Paul-Georg Germann
May 29, 2013·International Journal of Molecular Sciences·Manal FareaAdam Husein
Dec 30, 2003·Cytometry. Part B, Clinical Cytometry·M E J FranssenP E J Van Erp
Aug 13, 2005·Proteomics·Gilles LemaîtreGilles Waksman
Jul 8, 2005·International Immunology·Kavitha ViswanathanFirdaus S Dhabhar
Aug 4, 1998·The Prostate·R J BarrenG P Murphy
Mar 14, 2002·Toxicologic Pathology·Danielle RomanPaul-Georg Germann
Feb 12, 1998·The British Journal of Dermatology·C P GladeP C van de Kerkhof

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