Plan apochromat 60 1.4 oil dic n2 objective

Melanoma cells (1.25×10⁵) were seeded onto a cover glass placed inside a 24-well plate. Cells were left to adhere for 2 h, after which vemurafenib or trametinib was added for 12 h. Next, the culture medium was discarded, and cells were washed with PBS followed by fixation/permeabilization with methanol for 10 min. Cells were incubated in a blocking solution: 5% phosphoBLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05% for 1 h. Next, primary antibodies against phospho-H2AX (Ser139, #2577) (Cell Signaling Technology, Danvers, MA, USA) were added for 1 h, and cells were washed with PBS-Tween 0.05% followed by 1 h incubation with secondary, Alexa Fluor555-conjugated (#ab150078) antibodies from Abcam (Cambridge, UK). After washing with PBS-Tween 0.05%, the coverslips were flipped onto a drop of ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides and left to dry overnight. Images were obtained using the Nikon Eclipse TE2000-S inverted microscope (Nikon) with Plan-Apochromat 60×/1.4 Oil DIC N2 objective and Nikon C1 confocal attachment. Nuclear fluorescence was imaged with 408 nm excitation and 432–467 nm emission range, while secondary antibody fluorescence was imaged with 543 nm excitation and 567–642 nm emission range.

The hGC33-PE38 immunotoxin was covalently stained with the fluorescent dye ATTO 542 NHS-ester (Atto-Tec GmbH) according to manufacturer's protocol. NCI-H446 and NCI-H510A cells were incubated with 4.5 µg/ml ATTO 542-stained hGC33-PE38 immunotoxin in complete medium for 1 h at 37°C. Subsequently, cells were washed twice with complete medium for 10 min each and resuspended in HBSS with 1 µM calcein-AM (Thermo Fisher Scientific, Inc.) and 4 µM Hoechst 33342 (Thermo Fisher Scientific, Inc.). After another 20-min incubation at 37°C, a portion of cells were transferred onto the SensoPlate. The plate was centrifuged for 3 min at 300 × g at room temperature and the cells were then imaged. Images were obtained using the Nikon Eclipse TE2000-S inverted microscope with and Plan- Apochromat 60×/1.4 Oil DIC N2 objective and a Nikon C1 confocal attachment (all from Nikon Corporation).