Cell Motility

The automated screening of compounds for their effects on lateral cell motility is challenging because most assays either involve the direct manual observation of live cell movement by time-lapse microscopy which is time consuming and laborious, or by inference from more indirect invasion assays by the ability of cells to transit through a membrane in a Boyden chamber. An alternative offered by Thermo Fisher Scientific is an automated HCS assay that enables the observation and quantitation of cell motility in two dimensions in a 96-well fixed end-point format. This assay is both quantitative and automated, enabling the screening of compound libraries to evaluate their effects on cell motility. The fixed endpoint nature of the assay confers timing flexibility in conducting and reading the assay plates during the screen, without worrying about the state of the live cells.

This cell motility assay uses a Thermo Scientific Cellomics HCS Reagents for Cell Motility and the ArrayScan VTI HCS Reader with the Cell Motility BioApplication image analysis software. The Cell Motility HCS Reagents are easy-to-use and have been developed and optimized to produce robust, consistent quantification of cell motility when measured on the ArrayScan VTI HCS Reader and provide a functional assay that can predict the efficacy of potential drugs for a number of therapeutic applications.

The assay was developed using a “lawn” of fluorescent beads deposited on the bottom of microplate wells over which the live cells are plated. These beads are displaced (and/or phagocytosed) by motile cells, causing a series of beadless “tracks” whose size and shape can be quantified. To ensure accurate representation of cell tracks, the cells are counterstained, measured and correlated with individual tracks. The HCS Reagents for the assay include buffers needed for processing the assay, blue fluorescent microspheres, and rhodamine-conjugated phalloidin, which stains F-actin, to identify the cell cytoplasm. Prepared cells can be analyzed via standard fluorescence microscopy or using ArrayScan VTI HCS Reader with the Cell Motility BioApplication image analysis software module which provides quantitative measurements of track area, thus affording automated plate handling, focusing, cell image acquisition, analysis and quantification.

Principle of Thermo Scientific Cellomics Cell Motility Assay. (A) Schematic of cells (red), beads (blue layer) and protein substrate (gray layer); (B) Bead microlawn displacement and quantification of cell “tracks” by imaging. Images of tracks (outlined in blue) generated by L929 cells (outlined in green) stimulated by the presence of serum for 18 hours. Cells were prepared according to the Cell Motility HCS Reagent protocol. Images were acquired by the ArrayScan VTI HCS Reader, and the tracks and cells were identified and outlined by the Cell Motility BioApplication.

The Thermo Scientific Cellomics Cell Motility HCS Assay returns statistically useful results for assay durations between 3 and 22 hours. Longer assay times produce larger windows at the expense of more frequent crossing of individual cell tracks. The area of the tracks produced by bead displacement and phagocytosis during cell movement increases proportionally with time
during the first day. Within the observed time period, the rate of cell motility is constant and not reduced due to the progressive phagocytosis of beads.