Cell migration plays an important role in physiological processes such as wound healing while its deregulation contributes to the pathology of cancer cell invasion and metastasis. Agents that affect cell motility, either positively or negatively, have the potential to serve as therapeutics for wound healing or as antimetastatic agents, respectively.
Investigators engaged in high throughput screening (HTS) of compounds seek improved, high-density format assays that offer: complete adaptability for liquid-handling automation to facilitate ease of use and decrease hands-on time; the ability to view cells in real-time during experiments, a feature that is lacking in Boyden chamber-based assays where the membrane insert is an obstruction; and greater reproducibility of data than in scratch assays where wound sizes can be inconsistent and where cells and underlying extracellular matrix coating may also be damaged.
Platypus Technologies has developed the Oris™ Pro 384 Cell Migration Assay, available with either tissue culture treated or collagen I coated surfaces, which is fully automatable, suitable for real-time viewing, offers increased robustness over Boyden chamber and wound healing/scratch assays, and is compatible with high-content analysis (HCA) systems.
The Oris Pro 384 Cell Migration Assay is a 384-well high density cell exclusion assay useful for screening compound libraries (Figure 1). The 384-well assay plates are provided with spots of biocompatible gel (BCG) deposited in each well to exclude cells from adhering in the centers of the wells. The user seeds cells into the BCG-containing wells to set up the assay. The BCG is formulated to rapidly dissolve as the cells fall to the well bottom and begin to attach. Full dissolution of the BCG reveals a cell-free detection zone reproducibly positioned in the center of each well into which cells may then migrate.
SKOV-3 cells (human ovarian adenocarcinoma) were labeled with Vybrant® DiD cell-labeling solution. Cell seeding and compound addition were performed using a Tecan Freedom EVO 200 liquid handler equipped with a robotic manipulator arm (RoMa), a multichannel arm (MCA384), a liquid-handling arm (LiHa), and a Cytomat 6001 automated incubator. Prelabeled cells (12,500/well in 25 μL of medium containing heat inactivated serum) were seeded into an Oris Pro 384 Cell Migration Assay Tissue Culture treated plate from a stirred reservoir using the MCA384 with disposable tips at a low dispensing speed.
After a four-hour incubation period, Cytochalasin D (0–4 μM, final concentrations; n=24 for each) was added to the assay wells in 10 μL of serum-free medium. Premigration images were captured using an IsoCyte DL™ laser scanning cytometer equipped with 488 nm and 640 nm lasers.
Scanning was performed at a 5 μm x 5 μm resolution using a 660–680 BP filter and required approximately 8 minutes per plate. The assay plate was returned to the incubator for 16 hours to allow the cells to migrate, after which time post-migration images were captured. Image analysis was performed with BBIsoCyte 5.1 software to determine the area of the detection zone covered by migrating cells.