Pathway 855 bioimager system

The intracellular NO location was analyzed with the aid of a real-time living cell imaging system. The M. truncatula cell suspension (200 µl in five replicates) was incubated in 5–30 µM DAF-FM DA (Sigma-Aldrich) in 10 mM Hepes–KOH pH 7.4 for 30 or 60 min at 25 °C in the dark. After incubation and washing three times in 10 mM Hepes–KOH pH 7.4, 50 µl of the suspension was transferred onto a standard 96-well plate with 200 µl of 10 mM Hepes–KOH pH 7.4. Cell imaging was performed with the aid of a BD Pathway 855 Bioimager system using λ_Ex/Em = 495/515 nm.

To determine the levels of cytotoxicity caused by the experimental compounds (L-28, L-23, PMSF, GRK2i) previously described DNS assay adapted for high-throughput screening was used [36 ]. This assay uses two fluorescent nucleic acid intercalators, Hoechst 33342 (Hoechst) and propidium iodide (PI). Briefly, PC12 cells were seeded in a 96-well plate format and incubated with NGF and inhibitors. One h before image capturing, cells were added with a staining mixture of Hoechst and PI at a final concentration of 1 μg/mL for each dye. Subsequently, cells were imaged in live-cell mode using a BD Pathway 855 Bioimager system (BD Biosciences, Rockville, MD). Montages (2×2) from four adjacent image fields were captured per well in order to acquire an adequate number of cells for statistical analysis, utilizing a 10× objective. To determine the percentage of dead cells from each individual well, both image acquisition and data analysis were performed using the BD AttoVision v1.6.2 software (BD Biosciences), and each experimental condition was assessed in triplicate.