Glomax discover multimode detection system

Analyses of NER, as well as ICL repair capacity and complementation with WT full-length XPF or XPG, as well as the respective splice variants was assessed via HCR performed according to the descriptions in Lehmann et al. [14 (link)] after 48 h for XPF in immortalized MRC5Vi cells and 72 h for XPG in primary patients or WT fibroblasts. Liposomal transient transfections were carried out in a 24-well format (Greiner Bio-One, Kremsmünster, AUT) using Attractene transfection reagent (Qiagen, Hilden, DE) according to the manufacturer’s instructions. Unirradiated Renilla luciferase was used for normalization. Luminescence was measured 48 h or 72 h after transfection with the GloMax^® Discover Multimode Detection System (Promega). The relative repair capability [%] was calculated as the quotient of treated cells to untreated firefly/Renilla ratios. Cells were treated with either UVC (750–1000J/m²), with diammineplatinum(II) dichloride (CP) (Sigma-Aldrich, Munich, DE) in a molecular ratio of 1:20 or 1:40 (vector:CP) or with 4,5’,8-trimethylpsoralen (TMP) (Sigma Aldrich, Munich, DE) (1:25 (primary cells) or 1:50 (immortalized cells) (vector:TMP)) followed by irradiation with 1 J/cm² UVA. A batch treatment of plasmids was done and used for all respective experiments. The assay was repeated at least four times in triplicates.

Differentiated adipocytes were incubated in FBS-free DMEM mixed with 250 nM thermosensitive fluorescent ERthermAC dye for 30–45 min at 37 °C [34 (link)]. Media were replaced with fresh 90 μL DMEM/H without phenol red prior to imaging. Fluorescence in the stained cells was detected via a GloMax Discover Multimode Detection System (Promega, Madison, WI, USA) using 520 nm excitation and emission at 580–640 nm. The temperature inside the machine was equilibrated at 25 °C. After measuring 3 points of basal fluorescence, 10 μL forskolin (final concentration: 10 μM; F6886, Millipore Sigma, Burlington, MA, USA) was added to initiate thermogenesis. Fluorescence was recorded every 5 min over 120 min. Results were interpreted as relative intensity (normalized to basal measurements). The fluorescent intensity of the ERthermAC dye is inversely correlated with intracellular temperature. The area above the curve was quantified to show the thermogenic capacity in response to forskolin.

Cell viability was detected with Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instruction. Absorbance was measured at 450 nm using a GloMax Discover Multimode Detection System (Promega, Madison, WI). SC viability (%) was calculated using the following formula: $$\begin{array}{l}(\mathrm{absorbance}\mathrm{of}\mathrm{treatment}\mathrm{group}-\mathrm{absorbance}\mathrm{of}\mathrm{blank})/(\mathrm{absorbance}\mathrm{of}\mathrm{control}\mathrm{group}\\ -\mathrm{absorbance}\mathrm{of}\mathrm{blank})\times 100\%\end{array}$$
The live-cell growth status was recorded by photography using an IncuCyte Zoom System (Essen BioScience, Inc., Ann Arbor, MI).
Cell apoptosis was evaluated using annexin V/propidium iodide (PI) staining. SCs were washed with ice-cold phosphate buffered saline (PBS), centrifuged (100 × g) for 5 min at 4 °C, resuspended in 1× binding buffer of an Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (BD Biosciences, San Joes, CA). Five microliters of fluorescein isothiocyanate-labelled annexin V and 5 µl of PI were added to a 490 µl suspension and were gently mixed. After incubation at 25 °C for 15 min in the dark, SCs were analyzed using BD Accuri^TM C6 Flow Cytometer (BD Biosciences).