Arvo x

The relative cell number was determined by the measurement of DNA content in the well. At the cell cultures endpoints, the wells were emptied of media and rinsed once with PB containing 0.8% NaCl (PBS).
The wells were stored at -30°C. To perform the assay, the well plates were thawed and digested in a cell lysate buffer (0.1 mg/ml Proteinase K with 0.01% SDS in PBS). The DNA content of the sample was measured after appropriate dilution with PBS using a fluorescent plate reader (ARVO X, PerkinElmer, Waltham, MA) (excitation, 355 nm; emission, 460 nm) with the addition of Hoechst 33258 (2 μg/ml). DNA from salmon sperm was used as a standard.

SH-SY5Y cells were plated on 96-well plates at the density of 20,000 cells/well ( Fig. 4 C-F) or on 384-well plates at the density of 5,000 cells/well ( Fig. 4 G-I). After incubation as indicated, cell number was first estimated using CellTiter-Fluor TM Cell Viability Assay (G6080, Promega, Madison, WI, USA). CellTiter-Fluor Reagent was added to the cell culture medium at a 1:4 ratio, mixed, and incubated for 30 min at 37 °C. Fluorescence was detected GloMax® Discover Microplate Reader (GM3000, Promega, Madison, WI, USA) or ARVO X (PerkinElmer, Waltham, MA, USA), and cell density was obtained according to the manufactural instruction. Then, the Nano-Glo® Luciferase Assay System (N1110, Promega, Madison, WI, USA) was used to measure Nanoluc expression, carried out according to the manufacturer's instruction. Briefly, the Nano-Glo Luciferase assay reagent with the substrate was added to the cell culture medium at a 1:1 ratio. After 5 min incubation, luminescence was detected using the GloMax® Discover Microplate Reader, ARVO X (PerkinElmer, Waltham, MA, USA) or PHERAstar FSX (BMG LABTECH, Ortenberg, Germany). The obtained values of luminescence were normalized by the cell density, and relative luminescence was calculated relative to the control sample.