96 well microplate

BNP and ANP oligomers were generated by incubating the peptides at room temperature for 24 h, 3 days, and 7 days at a concentration of 30 μmol/l in PBS. Atrial HL-1 cells were plated at a density of 25,000 cells per 100 μl Claycomb Medium/well in a 96-well microplate (Perkin Elmer, Waltham, Massachusetts) pre-coated with gelatin and fibronectin, and incubated overnight (37°C, 5% CO₂). Cells were then treated with BNP and ANP oligomers (0.45 μmol/l) for 24 h. At the end of the treatment, cytotoxicity of BNP and ANP oligomers on HL-1 cells were determined by measuring cellular ATP levels with an ATPlite assay (Perkin Elmer) according to the manufacturer’s instructions. Luminescence was measured using a Lumicount microplate reader (Global Medical Instrumentation, Ramsey, Minnesota).

Cells were seeded in 96-well microplates (PerkinElmer); the seeding cell confluency was specifically optimized for each cancer cell line to have cells in a growth phase at the end of the assay. After overnight incubation at 37 °C, cells were treated with DMSO (Merck) for the negative control and with five concentrations of selected drugs in triplicate. Cells were then incubated at 37 °C for 72 h. Cell viability was assessed by measuring either luminescence with GloMax® Discover instrument from Promega or by nuclei count using the Operetta instrument from PerkinElmer. Luminescence measurements were normalized using background wells as manufacturer protocol. For luminescence measurement, cells were treated with Promega CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer protocol. For nuclei count, cells were washed with PBS 1×, fixed with paraformaldehyde (PFA) 4% for 10 min at room temperature, washed with PBS 1×, incubated at room temperature in the dark with HOECHST 33342 (Thermo Fisher Scientific) diluted 1:1000 in PBS 1× for 10 min and finally washed with PBS 1×. Nuclei count was performed using Columbus image analysis software (PerkinElmer). All drugs used in this study were purchased from Selleckchem.

In this study we used A. thaliana Columbia-0 (Col-0) transgenic lines carrying the following reporter genes in the Col-0 (or Col-5) genomic background: PR1p::GUS (Shapiro and Zhang, 2001 (link)), DR5::GUS (Ulmasov et al., 1997 (link)), WRKY29p::GUS (Serrano et al., 2007 (link)), and DC3::GUS (Chak et al., 2000 (link)). Arabidopsis seeds were surface-sterilized and seedlings grown hydroponically in 96-well microplates (PerkinElmer Inc., Germany) containing 0.2 ml of half-strength MS basal salt medium (Murashige and Skoog, 1962 (link)) supplemented with 0.5% sucrose. After stratification for 2 days at 4°C in the dark, plates were placed for 12 days in a growth chamber at a day/night cycle of 16/8 h at 21/19°C, respectively.

To measure luciferase activity in liquid culture, strains containing the luxCDAEB operon (under the control of the topA promoter or under the control of the erm promoter) in the pFLUXH ΦBT1 integrating vector were grown in liquid 79 medium for 24 h at 30 °C (in three biological replicates for each strain). Subsequently, the mycelium was collected by centrifugation, wet weight was determined, and mycelium was resuspended in 300 µl of 79 medium. Measurement of the luciferase activity was performed in triplicate directly from the mycelium suspension for each biological sample in a 100 µl volume in 96-well microplates (Perkin Elmer, Waltham, MA) using the Infinite PRO Multimode Plate Reader (Tecan, Männedorf, Switzerland). The luminescence intensity was normalized against wet weight (units/100 mg of mycelium). Luminescence visualization on solid medium was performed on MS agar plates after 48 h of growth at 30 °C, and luminescence detection was performed using a ChemiDocXRS + device (Bio-Rad, Hercules, CA).