Open access to high-content clonogenic analysis.
Image-processing programs are used to identify and classify eukaryotic cell colonies as spots following seeding at low density on dishes or in multiwell plates. The output from such approaches, however, is generally limited to 1-2 parameters, and there is no ability to extract phenotypic information at the single colony level. Furthermore, there is a lack of user-friendly pipelines for analysis of clonogenicity in the context of high-content analysis. This article describes an experimental and multiparametric image analysis workflow for clonogenic assays in multiwell format, named the Colony Assay Toolbox (CAT). CAT incorporates a cellular-level resolution of individual colonies and facilitates the extraction of phenotypic information, including the number and size of colonies and nuclei, as well as morphological parameters associated with each structure. Furthermore, the pipeline is capable of discriminating between colonies composed of senescent and nonsenescent cells. We demonstrate the accuracy and flexibility of CAT by interrogating the effects of 2 preclinical compounds, Nutlin-3a and ABT-737, on the growth of human osteosarcoma cells. CAT is accessible to virtually all laboratories because it uses common wide-field fluorescent microscopes, the open-source CellProfiler program for colony image analysis, and a single fluorescent dye for all the segmentation steps.
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia (IIT), Milan, Italy fernanda.ricci@iit.it mark.wade@iit.it.
Fernanda
F
Ricci
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia (IIT), Milan, Italy.
Aishwarya
A
Subramanian
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia (IIT), Milan, Italy.
Mark
M
Wade
D018929:Cell Culture Techniques; D002460:Cell Line; D003114:Colony-Forming Units Assay; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007093:Imidazoles / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D010879:Piperazines / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D057188:Workflow
D007093:Imidazoles; D010879:Piperazines; C482205:nutlin 3
CellProfiler; colonies; high content; p53; wide-field microscope