Incucyte flr imaging system

For protein stability assays cells were treated with 1mg/ml cycloheximide (Sigma, C7698-1G), 10 μM MG132 (Calbiochem, 474790) or 10 μM lactaystin (Enzo, BML-PI104-1000) for 8 h unless otherwise indicated. Before starvation with HBSS (Gibco, 14025-050), cells were washed for 4 times with PBS. Actinomycin D (Calbiochem, 114666) was used at a concentration of 1 μM. Cell viability was determined using an Incucyte FLR imaging system (Essen Bioscience, Ann Arbor, MI). Cells were plated in medium containing 30 nM SYTOX Green (Invitrogen, S7020). Cells were treated as described, imaged every 30 min over a period of 3 d, and analyzed using Incucyte image analysis software (Essen Bioscience). For quantification, the SytoxGreen fluorescence was normalized to the confluency factor of the respective well and the percentage of SYTOX Green-positive cells was calculated using the maximum SYTOX Green fluorescence at 100% cell death. Alternatively, cell viability was analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA). For this purpose, cells were harvested following 24 h of treatment as indicated and stained with Alexa Fluor 647-ANXA5/annexin V (BioLegend, 640911) and 1 μg/ml propidium iodide according to the manufacturer's protocols. Analysis was performed using Cellquest Pro software (BD Biosciences).

Unless stated otherwise, cells were seeded at 1,000 cells/well into flat-bottom 96-well plates (Greiner 655180). Edge wells were excluded and plates were left for 30 min at room temperature before transferring to an incubator. Plates for real-time analysis were transferred to the IncuCyte FLR imaging system (Essen BioScience) as previously described (10 (link)). Unless stated otherwise, compounds were added (5 μL/well) at two days post seeding, for five days. Compounds were reconstituted in dimethyl sulfoxide (DMSO). Plates were assayed for cell viability using the resazurin reduction assay and/or cell counts as described previously (10 (link)). The Spark 20M (TECAN) was used to quantify resazurin fluorescence following best practice procedures described previously (11 (link)). The ImageXpress system (Molecular Devices) was used to image Hoechst 33342 stained plates. CellProfiler (www.cellprofiler.org) was used to enumerate cell counts as previously described (12 (link)). Normalized cell viability was calculated as follows: sample/DMSO × 100. Relative half-inhibitory concentrations (EC₅₀) were calculated in Prism from the nonlinear regression algorithm of the normalized cell viability plotted against an 8-point compound dilution.