Fig 1.
High-content cell cycle analysis based on DNA content.
(A) Example images of HT29 cells with nuclei stained with Hoechst 33342 and then segmented with Harmony software using Find Nuclei Method B or Method M. Arrows indicate examples of incompletely segmented nuclei. (B) DNA content histograms of Hoechst 33342 stained HT29 cells, prior to application of the PhenoLOGIC algorithm, using Find Nuclei method B and M. Red dotted lines indicate the position of the G1 and G2 peak. (C) DNA content histograms obtained from HT29 cells showing gating to identify cell cycle distribution. (D) Cell cycle distribution for asynchronous HT29, SKOV-3 and U87MG cells determined from DNA content histograms. Values are the average of 8 technical replicates ± SD.
Fig 2.
Multiparametric analysis of cell cycle distribution.
(A) Example images demonstrating DNA staining with Hoechst 33342, EdU incorporation and pHH3 (S10) expression in untreated HT29 cells. (B) Plots of DNA content versus log EdU content and DNA content versus log mean pHH3 (S10) expression to determine the major cell cycle phases in asynchronous HT29 cells. (C) Cell cycle distribution for asynchronous HT29, SKOV-3 and U87MG cells determined from multiparametric high-content images. Values are the average of 8 technical replicates ± SD.
Table 1.
Agents used to validate image analysis methodology.
Fig 3.
Validation of image analysis protocol.
HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU and pHH3 (S10). (A) Example histograms following treatment with DMSO, etoposide, paclitaxel or VX680. Cell cycle distribution was determined using either DNA histogram analysis (B) or multiparametric analysis (C). (D) Time course and dose response of cell cycle changes determined using the multiparametric method for cells treated with camptothecin, gemcitabine or etoposide. (E) Proliferation index (EdU positive cells treated / EdU positive cells control) and cell number were determined from the images. Values are the average of 6 technical replicates.
Fig 4.
Determination of cell cycle phase of γH2AX positive cells following treatment with a cytotoxic DNA damaging agent.
HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU, pHH3 (S10) and pH2AX (S139). (A) Example images from HT29 cells treated with DMSO, camptothecin or etoposide demonstrating the presence of pH2AX (S139) positive (γH2AX) nuclei. (B) Quantification of the fraction of γH2AX positive nuclei following treatment with the various agents. (C) Cell cycle distribution of γH2AX positive nuclei following multiparametric analysis. Values are the average of 6 technical replicates ± SD.
Fig 5.
Estimation of S-phase Duration using EdU-BrdU Dual Pulse Labelling.
(A) Images of HT29 cells labelled with various combinations of EdU and BrdU demonstrating the selectivity of Click-iT chemistry for EdU and MoBU-1 antibody for BrdU. (B) Percentage of cells labelled with the various combinations of EdU and BrdU. Values are the average of 6 replicates ± SD. (C) Dot plot of Mean Nuclear EdU versus Mean Nuclear BrdU demonstrating the positioning of gates for the various cell populations.
Fig 6.
Determination of cellular quiescence using nuclear RNA staining.
HT29 cells growing in 10% FCS or in 0.2% FCS for 72 hours were fixed and stained with Pyronin Y or RNAselect for determination of RNA and total DNA content. Plots of total nuclear DNA content versus total nuclear RNA content.
Fig 7.
Determination of cellular quiescence using nuclear Ki67 expression levels.
HT29 cells growing in 10% FCS or in 0.2% FCS for 72 hours were fixed and stained for Ki67 and pHH3 (S10) expression, total EdU and total DNA content. (A) Example images demonstrating Ki67 staining in HT29 cells. (B) Plots of total DNA versus log mean Ki67 fluorescence intensity demonstrating the identification of the quiescent (Ki67 negative) cell population. (C) Cell cycle distribution of HT29 and U87MG cells grown in 10% or 0.2% FCS determined from multiparametric high-content images. Values are the average of 8 technical replicates ± SD.