Nucview caspase 3 detection reagent

Multicellular structures were double-stained with SYTO 62 fluorescent dye (Invitrogen) and NucView caspase-3 detection reagent (Essen Bioscience). 3D confocal images were acquired with a Zeiss Axiovert-200M microscope, equipped with Yokogawa CSU22 spinning disc confocal unit using Zeiss Plan-Neofluar 5x objective. Intensity projections were created with SlideBook (Intelligent Imaging Innovations Inc, Denver, CO, USA). Background noise was removed by normalization, using either SlideBook or ImageJ (NIH, Bethesda, MD, USA) programs. The AMIDA program can be freely downloaded and is also available as supplementary file (AMIDA Program S1). Also a collection of exemplary images used for analyses performed by AMIDA, as shown in this manuscript, is available as a supplementary data file (Image Data S1).

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.