Images was collected using the STACK assay detailed in Forcina et al. (2017) (link). Images were acquired using the IncuCyte S3 microscope (Essen Biosciences; 1408×1040 pixels, at 1.24 μm/pixel). Acquisition settings for the green channel were ex: 460 ± 20, em: 524 ± 20, acquisition time: 300ms; and red channel were ex:585 ± 20, em: 635 ± 70, acquisition time: 400ms. Imaging was performed using a 10x objective. For all experiments, on Day 0 just prior to drug addition, images were taken of a control plate treated with growth media containing 500 nM SYTOX Green as detailed above. For kinetic analysis, images were acquired every 6–8 hours for every well of each plate for 72 hours. For experiments where kinetic analysis was not used images were collected only at the 72 hour end point.
For some experiments that did not require kinetic analysis, images were acquired using an EVOS FL Auto 2 automated microscope (ThermoFisher Scientific). Images were acquired using a 10x objective (EVOS 10x objective, Cat #: AMEP4681). Sytox images were acquired using a GFP filter cube (EVOS LED Cube, GFP, Cat #: AMEP4651, ex: 470/22, em: 525/50, acquisition time: 13.5ms) Mkate2+ images were acquired using a TexasRed filter cube (EVOS LED Cube TxRed, Cat #: AMEP4655, ex: 585/29, em: 628/ 32, acquisition time: 642.0ms).
For some experiments that did not require kinetic analysis, images were acquired using an EVOS FL Auto 2 automated microscope (ThermoFisher Scientific). Images were acquired using a 10x objective (EVOS 10x objective, Cat #: AMEP4681). Sytox images were acquired using a GFP filter cube (EVOS LED Cube, GFP, Cat #: AMEP4651, ex: 470/22, em: 525/50, acquisition time: 13.5ms) Mkate2+ images were acquired using a TexasRed filter cube (EVOS LED Cube TxRed, Cat #: AMEP4655, ex: 585/29, em: 628/ 32, acquisition time: 642.0ms).