Lung cancer cell lines were treated with cisplatin for up to 24 h and fixed on Superfrost Gold Slides using ice-cold methanol. Cells were stained overnight at 4°C using a primary antibody that specifically recognizes CDDP-GpG DNA adducts (RC-18). Antibody binding was detected using an anti-rat Cy3^®-labelled antibody and counterstained using DAPI (1 μg/ml (w/v). Images were acquired on an Axioplan fluorescence microscope (A). The quantitative data for adduct levels (B) in all cell lines (MOR, H460, A549 and SKMES-1) were measured using the ACAS-6.0 image analysis system as described in the Materials & Methods section. This CCD camera-based methodology does not deliver photographic images but directly measures the area of individual nuclei, their DNA content and the levels of Pt adduct-derived fluorescence signals from automatically selected pixels. This is represented as relative numbers for all three parameters for 100–500 cells. These are then converted into AFU (arbitrary fluorescence units) values representing the relative adduct level of a single nucleus. The graphics represented in Figure 11B show mean values of approximately 200 nuclei per time point ±95% confidence intervals. In parallel with these analyses, cell images for all cell lines were acquired using Axioplan fluorescence microscopy.

https://doi.org/10.1371/journal.pone.0233739.g001