Luciferin

As shown in Fig. 1B, ATP measurements were performed at D0, D7 and Re-D7. At first, cell sheets gained by cultivation of OECs derived from luc tg rats were washed 3 times in HBSS, and incubated in HBSS for 1 hour at 4 °C. Then, beetle luciferin (Promega Corporation, USA) was added at a final concentration 0.19 mg/mL and photon intensity in the cell sheet was immediately measured before preservation based on the luciferase/luciferin reaction (to estimate the ATP levels) using a luminescence microplate reader (Centro LB960; Berthold Technologies GmbH & Co. KG. After each measurement, each cell sheet was washed 3 times in HBSS, immersed in the preservation medium, and left for 7 days at 4 °C.
The cell sheets were washed 3 times in HBSS after preservation, and the amount of ATP was measured (for comparison at D0 and D7). The medium was changed to KCM after ATP measurement at 7 days of preservation, and the sheets were re-cultivated for 7 days at 37 °C and 5% CO₂. After 7 days of re-cultivation, the amount of ATP was measured again. The ATP levels were calculated as the ratio (%) of ATP levels measured before preservation.

The luciferase activity of whole male flies on Luciferin (Gold-biotech) containing agar/sucrose medium (170 μl volume, 1% agar, 2% sucrose, 25 mM Luciferin), was monitored in Berthold LB960 plate reader (Berthold technologies, USE) in l-36LL Percival incubators with 90% humidity (Percival Scientific, Perry IA). Three flies per well were covered with needle-poked Pattern Adhesive PTFE Sealing Film (Analytical sales and services 961801). The distance between the agar and film was such that the flies were not able to move vertically. Period length was determined from light measurements taken during the first two days of DD. The analysis was limited to this window because TIM-LUC and BG-LUC oscillations severely dampened after the second day of DD. Period was estimated by an exponential dampened cosinor fit using the least squares method in MS Excel (Solver function).