Cells were seeded into 8-well chamber slides (Thermo Scientific, Rochester, NY, USA) at a concentration of 100,000 cells/400 µL/well in full medium, allowed to attach overnight, followed by serum-starvation (1% FBS) for another 24 hours and incubated for 24 and 48 hours with the test substances that were diluted in fresh serum-starved medium. The fluorescent LIVE/DEAD Cell Imaging Assay (488/570 nm; Lifetechnologies, Eugene, OR, USA) was performed following the manufacturer's instructions.68 –71 (link) Cells were visualized under 100x magnification using a fluorescent microscope (Leica DMI 6000B, Wetzlar, Germany) with a monochrome digital camera (DFC350FXR2; Leica) and the filter sets Cy5/Y3 (exposure time 1030 ms, intensity 3, and gain 6.1) and Alexa488/L5 (exposure time 545 ms, intensity 3, and gain 6.1). Negative controls were performed by microscoping chamber slides without addition of fluorescent solution.
Three digital images per well were randomly taken (Leica Application Suite Advanced Fluorescence software 2.7.0.9329), blinded, and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).72 (link) Cell survival was calculated by building a quotient out of the area of living cells and the total cell area for each image.
Three digital images per well were randomly taken (Leica Application Suite Advanced Fluorescence software 2.7.0.9329), blinded, and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).72 (link) Cell survival was calculated by building a quotient out of the area of living cells and the total cell area for each image.
