October 1, 2008 (Vol. 28, No. 17)
Keren I. Hulkower
Membrane-Free Assay Design Provides Detection Flexibility for Drug Screening
Dysregulated cell migration has been implicated in cancer, macular degeneration, and diabetic wound healing while cell invasion through an extracellular matrix is a hallmark of tumor cell metastasis. Understanding the processes involved in cell movement can provide important insight into the management of multiple disease states.
Basic research is being performed to elucidate signal transduction pathways and the effects of alternate matrices that regulate cell migration and invasion. Drug discovery efforts can be greatly augmented through the use of automation-friendly assays that provide more native environments to model in vivo conditions while permitting continuous visualization of cell motility not possible with trans-membrane cell culture inserts.
The reproducible, fluorescence-based cell motility assays demonstrated in this tutorial allow the use of multiple labels and stains to provide in-depth data and offer compatibility with diverse instrumentation platforms.
Real-Time Imaging and Quantification
A 96-well cell motility assay platform from Platypus Technologies utilizes cell-seeding stoppers to create a detection zone for visualizing cell migration and invasion as the assay progresses.
The Oris™ Cell Migration Assay features a 96-well plate coated with collagen, fibronectin, or a standard tissue culture-treated surface with silicone stoppers inserted in each well.
Adherent cells form an annular pattern around the stopper tip within a 4–18 hour period after seeding. Removal of the stopper reveals a pristine 2 mm detection zone in the center of the monolayer into which the cells can migrate (Figure 1).
A removable mask with 96 apertures is attached to the plate bottom permitting plate reader analysis. Use of GFP-labeled cells or fluorescent dyes allows analysis of cell migration throughout the experiment.
Cell movement may be detected by two methods: staining live cells (e.g., Calcein AM, CellTracker™ Green dye) or staining fixed cells (e.g., Hoechst, Alexa Fluor® phalloidin). Protein expression or morphology of fixed cells can be further elucidated by immunostaining to reveal additional data beyond migration.
The Oris Cell Invasion & Detection Assay is a true 3-D assay for studying cell invasion. In this assay, basement membrane extract (BME) is coated on the wells of the plate prior to stopper insertion. Cells are then seeded on the BME substrate, stoppers are removed to reveal the detection zone, and a second aliquot of BME is overlayed to encapsulate the cells. After the desired incubation time of 24–72 hours, cells are stained with Calcein AM to quantify the degree of invasion.
These assays have dramatically lower coefficients of variance (<20%) than wound-healing or scratch assays. HT-1080 cells were incubated for at least six hours in the Oris Cell Migration Assay then fluorescently stained and quantified using a BioTek Synergy™ microplate reader. This yielded a Z-factor of >0.5, making the assay suitable for drug screening applications.
The Oris assay platform serves as a more physiologically relevant model of cell motility for drug discovery by using matrices in a membrane-free 2-D and 3-D environment not possible with Boyden chamber-based assays. Results from two applications are highlighted in this article.
Cell Migration and Invasion
The study presented in Figure 2 shows HT-1080 fibrosarcoma cells responding to the actin-myosin inhibitor Blebbistatin. Cells were seeded at 50,000 cells/well on the Oris Cell Migration Assay plate and incubated overnight in media containing 10% FBS. The stoppers were removed and the media aspirated and replaced with media supplemented with FBS and Blebbistatin.
Cells were allowed to migrate for 17 hours. Following staining with Calcein AM, migration on the 96-well plate was quantified in fewer than 10 minutes on TTP Labtech’s Acumen® eX3 microplate cytometer. Representative images of the detection zone are shown for premigration controls and cells migrating in the presence or absence of Blebbistatin.
The Acumen eX3 instrument provided both fluorescent images of migrating cells (Figure 2A) as well as an EC50 calculation for the Blebbistatin dose response (Figure 2B). Imaging cell movement does not require that the detection mask be attached to the Oris plate.
The Oris Cell Invasion & Detection Assay was used to study the effect of Blebbistatin on the invasiveness of MDA-MB-231 breast cancer and NMuMG normal murine mammary gland cells in a 3-D environment. Both cell lines were seeded at 50,000 cells/well on BME-coated plates and incubated overnight. Stoppers were then removed, wells overlayed with 40 uL of BME, and cells allowed to invade for 48 hours.
The MDA-MB-231 cells were stained with Calcein AM while the NMuMG cells were fixed and stained with Alexa Fluor phalloidin to detect actin. Images were obtained by use of a Nikon TE300 inverted microscope with 4X and 10X objectives and captured with a Photometrics CCD camera. A portion of the MDA-MB-231 cells invaded upwardly into the BME overlay as observed by the intensity of the Calcein AM stain in cells at different focal points within the z-axis.
The NMuMG cells also moved through the BME into the detection zone, however there was less movement in the z-axis with these nonmalignant cells compared to the MDA-MB-231 cells. Treatment of the cells with 10 uM Blebbistatin was effective in dramatically reducing the amount of cellular invasion into the detection zone (Figure 3).
Oris cell-based assays have proven useful for researchers investigating signal transduction pathways for migration and invasion and as physiologically relevant models for drug screening. These assays offer new opportunities to observe the mechanism of action through secondary staining of intracellular targets performed in a multiplexed fashion on the same assay wells following completion of the primary motility assay.
Keren I. Hulkower, Ph.D. ([email protected]), is R&D team leader and Maria Perr is technical support manager for Oris cell-based assays at Platypus Technologies.