March 1, 2011 (Vol. 31, No. 5)
Thilo Riedl, Ph.D. Genmab Senior Scientist
Jean-François Têtu, Ph.D.
Keren I. Hulkower, Ph.D.
Automatable, High-Density Assay Offers Benefits Not Found with Boyden Chamber Products
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.
SKOV-3 Cell Migration
The Oris Pro 384 Cell Migration Assay was used to study motility of SKOV-3 ovarian cancer cells in response to the actin polymerization inhibitor, Cytochalasin D. Representative pre- and post-migration fluorescent images of the detection zone obtained using the Isocyte DL are shown for DiD-labeled cells migrating in the presence or absence of Cytochalasin D (Figure 2). The premigration area of the detection zone (i.e., the cell-free area) was calculated as 2 x 106 µm2 4 hours after cell seeding, with a nominal variance of ± 5% across all wells of the assay plate.
In the absence of Cytochalasin D, the SKOV-3 cells migrated into the detection zone and covered a 9 x 105 µm2 area, or about 45% area closure of the previously cell-free detection zone after a 16 hour incubation period. In contrast, cells treated with 0.06 µM Cytochalasin D only covered a 5.5 x 105 µm2 area of the detection zone, or about 28% area closure. The 4 µM concentration of Cytochalasin D completely prevented cell migration as indicated by essentially equivalent area closure in the pre- and post-migration wells (Figure 2A).
Cytochalasin D inhibited SKOV-3 cell migration in a dose-dependent fashion, with a calculated IC50 of 0.09 µM (Figure 2B). In this study, a Z´ factor of 0.59 was achieved, with a coefficient of variance of <12% in assay wells treated with vehicle only.
The uniformly sized area of the detection zones established at the onset of this assay and a Z´ factor of 0.59 are results that attest to the reproducible and robust assay performance for the Oris Pro 384 Cell Migration Assay with SKOV-3 cells. While the assay is simple to perform, for any given cell type, empiric determinations of key assay parameters should be conducted to achieve optimal results when performing the Oris Pro 384 Cell Migration Assay. These parameters include: extracellular matrix requirements (e.g. tissue culture treated or collagen I coated); cell seeding volume and density to ensure that the Detection Zone is initially free of cells; and extent of migration time to minimize coefficients of variance and maximize Z´ factors.
The Oris Pro 384 well high density cell migration assay is an attractive option for high-throughput screening and high-content analysis for modulators of cell migration.
Thilo Riedl, Ph.D., is senior scientist HTS, and Arnout Gerritsen is associate director assay and bioanalytical science at Genmab. Jean-François Têtu, Ph.D. (firstname.lastname@example.org), is sales manager of cells and cell-based assays at tebu-bio. Keren I. Hulkower, Ph.D., is technical and applications manager at Platypus Technologies.