Titramax 101 orbital shaker

All ELISAs were performed in transparent 96-well f-bottom high-binding polystyrene microplates from Greiner Bio-One (Frickenhausen, Germany). Wells were filled by use of Eppendorf Research^® pro multichannel pipettes and dilutions of components were made with Research^® plus piston stroke pipettes from Eppendorf (Hamburg, Germany). For incubation, microplates were sealed with Parafilm^® from Bemis (Neenah, WI, USA) and shaken on a Titramax 101 orbital shaker from Heidolph Instruments (Schwabach, Germany). Washing steps were performed on a Microplate Washer 405 LS from BioTek Instruments (Winooski, VT, USA) and absorbance measurements on a SpectraMax Plus 384 microplate reader from Molecular Devices (San José, CA, USA).
Pure water was taken from a Merck Millipore Milli-Q Reference water purification system. Weighing was performed on a Sartorius (Göttingen, Germany) Research R180D-*D1 analytical balance. Measurements of pH values were carried out with a SevenEasy pH meter S20 from Mettler Toledo (Columbus, OH, USA).

Cells were inoculated (20×10³) into 96-well plates and left to incubate in complete media containing 10% FBS for 24 h. Cells were then treated with media only or HYP (2 μM), in the presence of modulators (where appropriate) and irradiated. The cell viability was assessed by the MTT assay 24 h post-irradiation. The assay was performed by replacing cell media with complete media containing 0.5 mg/mL MTT and incubating at 37 °C in a 5% CO₂ humidified atmosphere for 3 h. MTT media were subsequently aspirated from all cells and the produced formazan crystals solubilized with 100 μL DMSO per well. The plates were shaken for 10 min at ~300 rpm in a Heidolph Titramax 101 orbital shaker (Heidolph Instruments GmbH & Co.KG), and the endpoint absorbance measurements at 570 nm were performed in a BioTek PowerWave XS2 plate reader (BioTek Instruments, Inc.). Blank values measured in wells with DMSO and no cells, were in all cases subtracted.