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.