title: Low-magnification image analysis of Giemsa stained, electroporation and bleomycin treated endothelial monolayers provides reliable monolayer integrity data.
abstract: The aim of this study was to develop an in vitro cell model for studying the in vivo observed vascular effect, induced by exposing blood vessels to changing electric field strengths. Human microvascular endothelial cells (HMEC-1) were cultured as monolayers on 8 chamber glass slides as a model of capillary wall. Exposed to electric pulses alone, or in the presence of bleomycin (electrochemotherapy), monolayers were incubated with culture medium, fixed with methanol, stained with Giemsa, and photographed. Images of high-contrast low-magnification monolayers made under identical optimal light exposure were converted to greyscale, and the use of a threshold tool yielded a binary distribution, from which we determined two parameters of monolayer integrity: the covered surface area and the number of cells. We show that this low-magnification image analysis method for attached endothelial cells provides reliable control parameters of monolayer integrity, representing capillary wall. Besides, already within 2h post-treatment the data show distinct effects in the monolayer integrity parameters for electric pulses alone, or in the presence of bleomycin. The present method can be readily introduced to short and long-term toxicity assays with a variety of treatment conditions.
authors.affiliation: Faculty of Health Sciences, University of Primorska, SI-6310 Izola, Slovenia. Electronic address: cecil.meulenberg@fvz.upr.si.
authors.forename: Cécil J W
authors.initials: CJ
authors.lastname: Meulenberg
authors.affiliation: Faculty of Health Sciences, University of Primorska, SI-6310 Izola, Slovenia; Institute of Oncology Ljubljana, SI-1000 Ljubljana, Slovenia.
authors.forename: Maja
authors.initials: M
authors.lastname: Cemazar
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001399:Azure Stains; D001761:Bleomycin / Q000494:pharmacology*; D002460:Cell Line; D018274:Electroporation; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects*; D006801:Humans; D015203:Reproducibility of Results
chemical_list: D000903:Antibiotics, Antineoplastic; D001399:Azure Stains; D001761:Bleomycin
keywords: Bleomycin toxicity; Cell viability; Electroporation; Microvascular endothelium; Monolayer integrity