Lb 940 multimode plate reader

To assess mitochondrial and glycolytic ATP generation, transfected (control and SIRT5 RNAi) hPTECs were either incubated with 25 mM 2-Deoxy-d-glucose in glucose-free DMEM supplemented with 5% FBS for 1 h or 2 µM oligomycin A in glucose-containing DMEM supplemented with 5% FBS for 25 min, respectively. The ATPlite Assay (Perkin Elmer) was performed according to the manufacturer’s protocol. ATP levels were normalized to cell number (CyQUANT; Life Technologies). Luminescence (ATP) and fluorescence (DNA) were measured with a Mithras LB 940 Multimode plate-reader (BERTHOLD).

Cultures were prepared and grown in NSMP medium supplemented with 0.1% (w/v) glucose as described for the luminometry experiment, with the differences that black plates (Nunclon Delta, Thermo Fisher Scientific) were used to reduce background signal. Cultures were exposed to light-darkness cycles for 3 days, after which the cultures were released to constant darkness conditions. The temperature was kept constant at 27°C. GFP fluorescence was red every hour using a multimode plate reader (Berthold Mithras LB 940 Multimode Plate Reader). All experiments were carried out in temperature-controlled incubators (Percival Intellus, Percival, USA). The plate was ejected from the machine between readings, for exposure to light. Signal from control wells (n = 23) with bacteria carrying no fluorescence reporter was subtracted as background.