Magellan v6

Corneal epithelium was scrapped with a dulled blade and nuclear protein extraction was performed according to the manufacturer's instructions. NF-κB p65 activation was measured by a TransAM NF-κB p65 kit that specifically quantifies phosphorylated NF-κB p65 (Active Motif, Carlsbad, CA, cat. no. 40596). Nuclear extracts from non-stressed and DS1 corneas were added to wells of a 96-well plate with immobilized oligonucleotide containing an NF-κB consensus binding site. The activated p65 in the nuclear extract binds to the oligonucleotide. After incubation with specific anti-p-p65 antibodies, horseradish peroxidase conjugated secondary antibodies provided a sensitive colorimetric readout at 450 nm using a colorimetric plate reader (Tecan Infinite M200, Magellan V6.55 software; Tecan, Männedorf, Switzerland).

The activation of caspase-3 was measured in lacrimal gland lysates according to the manufacturer’s protocol (K105-200, BioVision, Inc., Mountain View, CA, USA) [31 (link)]. Protein concentration was measured using a micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Five to nine samples per group were used. Caspase-3 activities were measured (50 μg/sample) by following the cleavage of the fluorescent substrate analogs in a fluorescent plate reader (Tecan Infinite M200, Magellan V6.55 software; Tecan, Männedorf, Switzerland) with 400-nm excitation filter and 505-nm emission filter. The results were exported and averaged.

Cells were fixed with ice-cold ethanol, washed with Aq_dest, and incubated with 40 mM Alizarin red solution (Sigma Aldrich, pH 4.1) for 20 min at RT. After thorough washing, the air-dried specimens were evaluated with a SZH10 microscope (Olympus, Münster, Germany) equipped with a CCD Colour view III camera and the resulting images were taken and analyzed using the CellSens software version 1.5 (both Olympus, Münster, Germany). For quantification, Alizarin red was extracted with 10% acetic acid for 30 min at RT. The cell layer was scraped, and the solution was covered with mineral oil (Sigma Aldrich) and incubated for 10 min at 85 °C. The supernatant was transferred into microplates, the absorbance was read at 420 nm in triplicates, and data were collected and analyzed using the Magellan v6.2 software (Tecan, Crailsheim, Austria).