Operetta harmony high throughput imaging platform

Plate and liquid handling was performed using a HTS platform system composed of a Sciclone G3 Liquid Handler from PerkinElmer (Waltham, MA, USA) with a Mitsubishi robotic arm (Mitsubishi Electric, RV-3S11), a MultiFlo^TM Dispenser (Biotek Instruments, Bad Friedrichshall, Germany) and a Cytomat^TM Incubator (Thermo Fisher Scientific, Waltham, MA, USA). Cell seeding and assays were performed in black 384-well CellCarrier^TM plates (PerkinElmer, 6007558). The diverse small molecule library used in HTS was acquired from Prestwick Chemical (Illkirch, France). We tested 960 small molecule compounds of the Prestwick Chemical library, which are 100% approved drugs (Food and Drug Administration (FDA), European Medicines Agency (EMA) and other agencies). The purity of the compounds was >90% as reported by the provider of the compounds. Cells were seeded 40h before treatment in 384-well microplates with a cell number of 4 × 10⁴ cells/well. This resulted in about 80–90% confluent after 16 h and in a confluent layer after serum starvation for another 24 h (time of compound addition). Image acquisition and image-based quantification was performed using the Operetta^®/Harmony^® high-throughput imaging platform (PerkinElmer, USA).

Plate and liquid handling was performed using an HTS platform system composed of a Sciclone G3 liquid handler from PerkinElmer with a Mitsubishi robotic arm (Mitsubishi Electric, RV-3S11), a MultiFlo dispenser (Biotek Instruments) as well as a Cytomat incubator (Thermo Fisher Scientific). Cell seeding and assays were performed in black 384-well CellCarrier plates (PerkinElmer, 6007558). The plates were coated with gelatin 0.1% for 20 min at 37 °C to facilitate better cell adherence. Cells were seeded in 384-well microplates with 10,000 cells per well. Image acquisition and image-based quantification was done using the Operetta/Harmony high-throughput imaging platform (PerkinElmer). Z′ factors were calculated according to the formula Z′ = 1 − (3(θ_p + θ_n)/(µ_p − µ_n)), where p is the positive control, n is the negative control, θ is the standard deviation and µ is the mean.