Low-magnification image analysis of Giemsa stained, electroporation and bleomycin treated endothelial monolayers provides reliable monolayer integrity data

•Microvascular endothelial monolayers used as an in vitro model of capillary wall.•Image analysis of low-magnification Giemsa stained endothelial monolayers.•Monolayer integrity represented by covered surface area and number of cells.•Exposure of monolayers to electroporating pulses, also in the presence of bleomycin.•More pronounced monolayer damage after electroporation in the presence of bleomycin.

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 2 h 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.