Harmony image analysis software

Adult human aortic smooth muscle cells (Thermo Fisher Scientific Inc.) were seeded at a density of 20,000 cells per well. The next day, HUVECs were plated at density of 5000 cells per well on the confluent smooth muscle monolayer in a 96-well plate as previously described [51 (link)]. GSK429486 (5 μM) or DMSO as a control was added one day after the plating of HUVECs. The co-cultures were sustained under full growth media (EGM-2) for seven days. Endothelial tubes were visualized via immunofluorescence using a mouse anti-human CD31 monoclonal antibody (R&D systems) at 1:400 dilution and goat Alexa 488-conjugated anti-mouse IgG secondary antibody (Thermo Fisher Scientific Inc.) at 1:500 dilution. Actin fibers were stained with Alexa 568-conjugated Phalloidin (Thermo Fisher Scientific Inc.). Nuclei were visualized via DAPI staining. The endothelial tubes were imaged automatically at 10× magnification using the high-content imaging system (Operetta). Nine identically positioned fields were acquired from each well in each experiment. The following quantification of endothelial network features, total tube length, branching points and mesh number, was performed automatically using an NIH Image J-based tool (angiogenesis analyzer) [94 (link)]. Meanwhile, the tube thickness feature was quantified automatically using Harmony image analysis software (Perkin Elmer).

HUVECs were pre-incubated with GSK429286 (10 μM) in a 96-well plate overnight. Next, cell permeable fluorescent Nitric Oxide (NO) indicator, 4-amino-5-methylamino-2′, 7′-difluorescein diacetate “DAF-FM-DA” (Thermo Fisher Scientific Inc.) was added at a final concentration of 2 µM for 30 minutes, as previously reported [99 (link),100 (link)]. Next, cells were washed, and NO production was visualized at excitation 460/490 and emission 500/550 using a high content fluorescence imaging system (Operetta). Intracellular fluorescence intensity was quantified in nine fields of each well using Harmony image analysis software (Perkin Elmer). Mean intracellular fluorescence was calculated by subtracting the integrated fluorescence density value obtained for the cell and the mean fluorescence of background readings.

Cells were transferred into CellCarrier 384-well plates (PerkinElmer, Waltham, MA, USA) at a density of 1250 cells/well, using Multidrop dispenser (ThermoFisher Scientific, Waltham, MA, USA). After overnight incubation at 37°C experimental, compounds were added with an ATS Acoustic Transfer System (EDC Biosystems, Fremont, CA, USA). For Ep156T cells the protocol was done in reverse with compounds dispensed before seeding of cells. Fluorescent markers were dispensed in culture medium using ATS system after 72-h compound exposure. Nuclei were stained with cell-permeable Hoechst 33342 (Molecular Probes, Eugene, OR, USA), dead cells with ethidium homodimer-2 (Invitrogen, Carlsbad, CA, USA) and apoptotic cells with NucView caspase-3 detection reagent (Essen Bioscience, Ann Arbor, MI, USA). Cells were imaged with Operetta high-content imager (PerkinElmer, Waltham, MA, USA). Proliferation was measured from the number of nuclei (cells), cell death and apoptosis from positive cells/total cells ratio using Harmony image analysis software (PerkinElmer, Waltham, MA, USA). EC₅₀ values were also assessed with the Harmony software.