of CFP/YFP, CFP was excited with 436610 nm band pass filter and CFP emission was collected using 483622 nm band pass filter. FRET channel was collected using 542627 nm band pass filter. Detection of FRET by flow cytometry CFP/YFP ratio analysis by FACS was performed in a FACSAria II (BD Biosciences) equipped with a 407 nm laser. Briefly, cells seeded in 12-well plates were treated with drugs. At the end of drug treatment, cell were trypsinized, washed twice with PBS and passed through a 40 mm sieve (BD Biosciences) before FACS analysis. To measure ECFP, cells were excited with the 407 nm laser and fluorescence was collected in the ECFP channel with the standard 450640 nm filter, while the EYFP FRET signal (for measurement of Venus) was measured with a 530630 nm filter. For each sample, we evaluated a minimum of 20,000 cells that fell within the background adjusted gate. In the scatter plot of ECFP Vs EYFP, the events that were detected within the gate drawn corresponding to increased ECFP and reduced EYFP were taken as cells with loss of FRET. Live cell staining Hoechst 33342 staining. Following drug treatment, treated and untreated cells were stained with 2 mg/ml Hoechst 33342 (Molecular Probes #H1399) in phenol-red free DMEM containing 5% FBS by incubating at 37uC for 10 minutes. Unbound dye was washed off by gently washing twice with PBS. Hoechst 33342 intensity was imaged for detecting pycnotic nuclei. Cells were counterstained with TMRM (Molecular Probes #T- 668) or MitoSOXTM Red (Molecular Probes #M36008) after Hoechst staining. TMRM staining: For TMRM staining, cells were incubated with 150 nM TMRM at 37uC for 10 mins following which, unbound dye was washed off. Stained cells were replenished with 5 nM TMRM containing 5% phenol red-free DMEM for imaging MMP loss. MitoSOX Red staining: MitoSOXTM Red mitochondrial superoxide indicator is a fluorigenic dye for highly selective detection of superoxide in the mitochondria of live cells. For staining, cells were loaded with 5% DMEM containing 5 mM MitoSOXTM Red and incubated for 10 minutes at 37uC, protected from light. Cells were washed thrice gently with PBS to remove excess unbound dye and replaced with fresh 5% phenol-red free DMEM. Highthroughput screening: acquisition, segmentation and analysis Image acquisition. For HTS imaging, cells were seeded in 96 well glass bottom plates (Greiner Bio-One) at desired density. After an overnight culture, medium was removed and replenished with phenol-red free DMEM containing 5% FBS with the compounds to be tested at the appropriate working concentrations. Plates were imaged under BD PathwayTM 435 Bioimager (Becton Dickinson Biosciences, USA). The filter combination for CFP/YFP FRET was designed as described for microscopic imaging with independent automated control of excitation, emission and dichroic filter wheels for easy adaptation of CFP/YFP ratio image acquisition with other parameters of choice. The excitation/dichroic mirror/emission filter combinations used for imaging were 438624/458LP/483632, 438624/458LP/542627, 562640/593LP/624640 and 377650/ 409LP/435LP for CFP, CFP-YFP FRET, TMRM/MitoSOX and Hoechst channels respectively for multiparmater high throughput image acquisition. Images for each well were acquired in the respective channels using a dry 206 objective with 0.75. Images were captured as single montage/363/666 montages. For caspase High Content Ratio Imaging for Apoptosis Detection inhibition, cells were pretreated for 1.5 hours with the respective caspase inhibitors Caspase inhibitor VI, Caspase 8 inhibitor I and Caspase 9 inhibitor II (Calbiochem) for inihibiting caspase 3, 8 and 9 activities respectively, following which, cells were subjected to drug treatment in the following manner: MCF7 SCAT3 and SCAT9 cells with Staurosporine (250 nM) and SCAT8 cells with Anisomycin (2 mg/ml) for 10 hours. At the end of the incubation time period, cells were subject to imaging for FRET loss positive cells. Post acquisition segmentation and analysis. Post- acquisition image analysis was done using BD AttoVisionTM (Version 1.6/435, Becton Dickinson Biosciences, USA) by applying simple polygon segmentation or polygon band segmentation for demarcating cytoplasmic and nuclear regions separately at the automatic threshold level and variable dilation as well as erosion width. Optimized segmentation masks were created for each experiment by an in-built distance-map based split algorithm for separating touching cells with desired erosion factor based on the cell type. Data was read out in the form of images and dot plots, with FRET ratio loss (increased DEVDase activity) corresponding to increased ECFP/EYFP ratio signal, TMRM intensity loss for mitochondrial membrane potential (MMP) loss, increase in Hoechst 33342 intensity for chromatin condensation and MitoSOX intensity gain for mitochondrial superoxide generation. Data export and quantitation. Following automated segmentation and ROI identification, the data was exported to BD FacsDivaTM and gates were applied for the corresponding quantitative read-outs. Gates were made in each experiment keeping basal apoptosis parameter in untreated controls below 10%. Read out of cells with greater FRET loss and chromatin condensation than the set threshold was taken as events in the upper quadrant, whereas, for MMP loss, those in the lower quadrant were considered. For the generation of intensity maps and Z’ factor values, data was exported to BD Image Data ExplorerTM (Version 2.2.15, BD Biosciences, USA). In order to estimate the ‘screenability’ of the HCS assay, Z’ factor values were calculated using the equation Z’ = 12(3*StdDevsample+3*StdDevcontrol)/ (Meansample2Meancontrol). SDS-PAGE and Western blotting 50 mg protein sample was resolved electrophoretically in 12% SDS–polyacrylamide gel and electro transferred onto a Hybond CTM extra nitrocellulose membrane (Amersham Biosciences) by wet transfer procedure (Bio-Rad Mini Protean11). The mem- brane, after blocking with 5% BSA in TBST was incubated with primary antibody overnight at 4uC. The primary antibodies used were: rabbit anti-GFP (#2555, Cell Signaling Technology), mouse anti-caspase 8 (#9746, Cell Signaling Technology), mouse anti- caspase 3 (#9662, Cell Signaling Technology), caspase 9 (#551247, BD Pharmingen), Cleaved caspase 3 (#9661, Cell Signaling Technology) and mouse anti-b actin (#A5316, Sigma) at 1:1000 dilutions. The membranes were then incubated in horse- radish peroxidase labeled anti-rabbit or anti-mouse secondary antibodies (Sigma) at 1:4000 dilutions for 1 hr and developed using Enhanced Chemi Luminescence assay (AmershamTM ECL Plus western blotting detection kit, GE healthcare, Buckingham- shire, UK) or DAB (Sigma). Results Validation of the caspase sensor transfected stable clones for presence and functionality of caspase sensor probe The image-based high content screening (HCS) methodology described here utilizes intracellular FRET-based caspase activity PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20114