High Content Ratio Imaging for Apoptosis Detection Figure 3. A representative output data with multiparmater analysis. A representative output data from OVCAR8 SCAT3 cells showing FRET loss with increase in donor blue fluorescence upon drug treatment in the CFP/YFP merge channels (B,I) with the corresponding FRET ratio pseudo- color channel (A, H) TMRM (D,K) and Hoechst fluorescence channels (C,J) (untreated: A–E, Camptothecin (10 mM, 12 hrs) treated H–L). Segmentaion mask (polygon band) showing spatial demarcation between nucleus and cytoplasm for both untreated (F&G) and camptothecin (10 mM, 12 hrs) treated (M&N) are also shown. The corresponding post-analysis quantitation carried out by FACS Diva software after indicated treatment is shown (O–R). Percentage of desired apoptosis parameter are shown in the corresponding gated areas FRET loss (O), hoechst intensity (P) and TMRM intensity (Q). Dot plot showing biparameter analysis of TMRM intensity Vs FRET loss (R). doi:10.1371/journal.pone.0020114.g003 treatment in the first round of treatment with 3 classes of drugs viz HDAC inhibitors (TriA, SBHA), translation inhibitors (Aniso, CHX) and microtubule inhibitors (Podo, Colchi) (Figure 10 i). When these surviving cells were subjected to a second round of treatment with the same drugs, we found that they took a longer time to respond, and showed a divergent caspase 9 activation response. At 24 hrs post drug treatment, cells that survived after treatment with microtubule inhibitors remained more resistant to a second phase of drug treatment; compared to HDAC inhibitors and translation inhibitors. For comparing death reponse, we scored for the percentage of cells that lost normal growing adherent morphology (rounded cells), treating these as dead cells (Figure 10 ii). The population that survived the first round of drug treatment showed a significant reduction in the percentage of dead cells at 24 hrs when compared to the same period of drug treatment of the parental population. The escaped population also exhibited a lesser level of caspase 9 activation in the rounded cells. This is clearly evidenced in the shown FRET ratio heat images by the absence of high FRET loss positive (orange) rounded cells in the escaped drug treated population, when correlated with the representative qualitative heat map. Discussion One of the earliest reports on the potential of GFP-FRET based caspase activation detection for anticancer drug screening in live cells came out in 2000 [26] with several improvisations of this method in the coming years such as advancements in fluorescence signal strength, stability and sensitivity [29–31]. Since the readout is from these assays are from well based integrated signal after meticulous background correction, they have limitation in multi-parameter pathway defining or deriving the single cell information. The HTS screening platform described here has several advantages starting from improved stability of probes used to adaptability for high content multiparameter apoptosis analysis in live cell mode. In compar- PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20114