Figure 1.

Multiparameter, high-content screening for compounds that promote β-cell survival. MIN6 cells stably expressing the caspase-3 eBFP-DEVD-eGFP FRET sensor under the Ins1 promoter were seeded into 96-well plates and stained with Hoechst 33342, PI, and Alexa Fluor 647–conjugated annexin V. Cell death was induced with a cytokine cocktail of 25 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ, and cells were treated with an 8.5 μM concentration of each compound in the Prestwick Chemical Library, which includes US Food and Drug Administration–approved drugs. Cells were imaged after 30 hours of treatment with ImageXpress Micro, and images were analyzed using MetaXpress software. A–D, Z score values calculated from the numbers of total cells, PI-positive cells, annexin V–positive cells, and activated caspase-3–positive cells were determined and displayed as a heatmap where red and green represent high and low numbers, respectively. The top hits that promoted β-cell survival were selected from automated clustering into 20 different self-organizing maps (A). Scatter plots of PI (B), annexin V (C), and caspase-3 (D) vs total cell were generated. CBZ, carbamazepine.