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Molecular analysis of rare single cells like circulating tumor cells (CTCs) from whole blood patient samples bears multiple challenges. One of those challenges is the efficient and ideally loss-free isolation of CTCs over contaminating white and red blood cells. While there is a multitude of commercial and non-commercial systems available for the enrichment of CTCs their cell output does not deliver the purity most molecular analysis methods require. Here we describe the ALS CellCelector™ which can solve this challenge allowing the retrieval of 100% pure single CTCs from blood processed by different upstream enrichment techniques. It is a multifunctional, extremely flexible system for automated screening of cell culture plates, Petri dishes, and microscope slides. Fixed or live single cells or multicellular clusters detected during screening can be picked out of those plates automatically. The complete scan and picking process is fully documented hence allowing highest standardization and reproducibility of all processes. Use of CellCelector allowed the isolation of pure single tumor cells or clusters from liquid biopsies of breast, prostate, ovarian, colorectal, lung, and brain cancers for their subsequent molecular analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2 .To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

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