Abstract
BackgroundCell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.ResultsWe have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.ConclusionsThe adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.
Highlights
Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes
To perform a wound healing assay, a wound is typically introduced in a cell monolayer using an object such as a pipette tip or syringe needle and the assay is performed on an individual coverslip or in a multiwell plate
In order to perform high throughput screening of cell migration, we developed a wound healing assay in a 384 well plate format that does not require expensive reagents, provides consistently shaped wounds, can provide detailed information on numerous processes involved in cell migration, and provides a quantitative, informationrich readout
Summary
Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. The wound heals in a stereotyped fashion – cells polarize toward the wound, initiate protrusion, migrate, and close the wound. Progression of these events can be monitored by manually imaging samples fixed at timepoints or by time-lapse microscopy. Wound healing assays are a classic and commonly used method for studying cell migration and the biology underlying it [4]. They have been used with multiple cell types and, as the monolayers heal the wound in a characteristic manner, they have been used to study cell polarization, matrix remodeling, cell migration, and numerous other processes [5,6,7]. The assay has been used as a proxy for angiogenesis, metastasis, and other physiological and pathophysiological processes [19,20,21,22,23,24]
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