Modulus 2 microplate multimode reader

The Shh, Fgfr2, and Fgf8 promoter fragment (0 to −2000 bp) was cloned from mouse genomic DNA and inserted into the pGL3.0 vector (E1751, Promega). Isl1 was amplified using the primers containing the NheI–XhoI restriction sites and inserted into the pcDNA3.1 vector. An empty luciferase reporter vector was used as a control. The primer sequence is in Supplementary Table S3.
The human embryonic kidney 293FT cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C with 5% CO₂. Cells were transfected with Isl1 expression vector, Fgf8, Fgfr2, Shh luciferase reporter vector, and pTK-Ranilla vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Twenty four hours post transfection, luciferase activity was measured by using Dual-Luciferase Reporter Assay Kit (E1910; Promega) on a Modulus II Microplate Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA). The values were normalized against Renilla luciferase activity. At least four independent experiments were performed.

Calcium mobilization in LAD2 cells was followed by fluorimetric analysis of cytoplasmic-free calcium with Fluo-4 AM fluorescent dye (Molecular Probes, Invitrogen) as described elsewhere [36] (link). Briefly, 0.2×10⁶ cells/point were loaded with 5 mM Fluo-4-AM for 30 minutes at 37°C in the dark, washed twice with Tyrode’s buffer, and resuspended. To measure calcium influx in the absence of extracellular calcium, cells were washed and resuspended with Tyrode’s buffer without calcium. Fluorimetric measurements were by a Modulus II Microplate Multimode Reader (Turner Biosystems, Promega, CA), according to the manufacturer’s instructions. After defining basal conditions the stimuli was added (time 0) and fluorimetric measures were done 10 more minutes. Each point was done by triplicate.

Cytotoxicity assay was performed using CellTox™ Green Cytotoxicity assay kit (Promega, Mannheim, Germany). Briefly, cells were cultured in 96-well plates at the density of 25 × 10³ cells/well in DMEM medium containing 10% fetal calf serum (Biochrom AG, Berlin, Germany) and antibiotics (40 U/mL penicillin and 40 μg/mL streptomycin, both from PAA Laboratories, Linz, Austria). Cells were pre-treated with different concentrations of AM404 (0.1–10 μM) or DMSO 0.1% for 30 min. Thereafter, cells were incubated with or without LPS for the next 24 h. Ethanol (10% end conc., Sigma-Aldrich, Taufkirchen, Germany) was used as positive control to induce the cell death. After incubation (10% CO₂ at 37 °C - Heracell 240i, Thermo Scientific), 100 μl of CellTox™ Green reagent were added in each well. The plate was mixed for 1 min and incubated for 15 min at room temperature, and the fluorescence was measured at 490 nm_Ex/530 nm_Em using a Modulus™ II Microplate Multimode Reader (Turner BioSystems, USA).
The principle of the assay is to evaluate the alterations in the membrane integrity, using the cyanine dye. The dye binds in the dead-cell DNA and enhanced the fluorescent property, which is excluded from viable cells. The fluorescence intensity values obtained were normalized and presented as the percentage of untreated controls.