Readyprobes cell viability imaging kit blue red

Cells were seeded at 2 × 10⁵ per well in a 12-well glass bottom plate (MatTek, Ashland, MA, USA). Detection of apoptotic cells was performed using the CellEvent™ Caspase 3/7 Green Detection Reagent (Life Technologies). After treatment, cells were stained with 50 µl CellEvent Caspase-3/7 green ready probes reagent and 50 µl ReadyProbes Cell Viability Imaging Kit Blue/Red (Life Technologies) for 15 min at room temp. z-stack images of stained cells were taken by confocal microscopy using a Nikon A1 confocal microscope with 20× objective. Active caspase-3/7: green fluorescence, Propidium iodide: red fluorescence, Nuclear DNA: blue fluorescence.

For assays, organoids were seeded as single cells in 40 μL of 33% Matrigel (mouse organoids) or Prostate 18 QGel 3D Matrix (NCI-H660 organoids) in 96-well tissue culture plates and cultured for 2 days at 37°C to allow organoid formation. Once formed, organoids were treated with AD80 (at concentrations of ranging from 0.1 μM to 30 μM), LOXO-292 or BLU-667 (at concentrations ranging from 0.01μM to 100μM), or 10 μM Enzalutamide (MedchemExpress) for 72 hours. After treatment, cells were stained with 10μL ReadyProbes Cell Viability Imaging Kit Blue/Red (Invitrogen) per well for 30 minutes at room temperature and z-stack images of stained cells were taken using an EVOS FL Auto 2 Cell Imaging System (Invitrogen). The percentage of cell death was calculated by identifying the percentage of PI-positive cells per organoid in at least 10 organoids for each treatment condition and the LD₅₀ was calculated in GraphPad Prism 7.

Due the Eosin Y autofluorescence in the green channel, cell survival was determined using ReadyProbes cell viability imaging kit, blue/red (Invitrogen). For each condition, 50 islets were dissociated with 0.05% trypsin-EDTA, filtered, and the resulting cell suspension was mixed with 80 μL of MAP gel or 80 μL of media and transferred to a 24 transwell plate insert. MAP scaffolds were annealed with 505 nm LED light (ThorLabs, 104 mW/cm² intensity) for 2 minutes. Wells received 1 mL of DMEM media and the plate was incubated at 37°C in between timepoints. After a 24-hour incubation, 2 drops of NucBlue Live reagent (Hoechst 33342) and 2 drops of propidium iodide were added directly to the well. After incubation for 10 minutes at room temperature, the transwells were imaged with 20X objective (1.4 mm correction collar) on a Molecular Devices ImageXpress Micro confocal microscope. Images were imported into ImageJ, and the red (dead) and blue (live) channels were thresholded and cells were counted using the ImageJ Particle Analysis plugin. Cell viability is reported as [(sum of live cells) / (sum of live + dead cells)]×100%.