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Cell Migration Assay for HTS and HCA

Automatable, High-Density Assay Offers Benefits Not Found with Boyden Chamber Products

• Thilo Riedl, Ph.D.
• ,
• Arnout Gerritsen
• ,
• Jean-François Têtu, Ph.D.
• ,
• Keren I. Hulkower, Ph.D.

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SKOV-3 Cell Migration

Figure 2. Effect of the actin-polymerization inhibitor Cytochalasin D on SKOV-3 cell migration using the Oris Pro 384 Cell Migration Assay. DiD-labeled SKOV-3 cells were seeded into an Oris Pro 384 using a Tecan Freedom EVO 200 liquid handler. (A) Pre-migration images, 4 hours after seeding, captured using IsoCyte DL laser scanning cytometer and analyzed with BBIsocyte 5.1 software. The premigration area of the Detection Zone was calculated as 2 x 106 µm2 ± 5% 4 hours after cell seeding. Cytochalasin D was added. Post-migration images were captured after an additional 16 hours. In the absence of Cytochalasin D, SKOV-3 cells migrated and covered a 9 x 105 µm2 area of the Detection Zone, corresponding to about 45% of the available area. Migrating cells treated with 0.06 µM Cytochalasin D covered a 5.5 x 105 µm2 area or about 28% area closure, while 4 µM Cytochalasin D completely prevented cell migration. Click Image To Enlarge +

Figure 2. Effect of the actin-polymerization inhibitor Cytochalasin D on SKOV-3 cell migration using the Oris Pro 384 Cell Migration Assay. DiD-labeled SKOV-3 cells were seeded into an Oris Pro 384 using a Tecan Freedom EVO 200 liquid handler. (A) Pre-migration images, 4 hours after seeding, captured using IsoCyte DL laser scanning cytometer and analyzed with BBIsocyte 5.1 software. The premigration area of the Detection Zone was calculated as 2 x 106 µm2 ± 5% 4 hours after cell seeding. Cytochalasin D was added. Post-migration images were captured after an additional 16 hours. In the absence of Cytochalasin D, SKOV-3 cells migrated and covered a 9 x 105 µm2 area of the Detection Zone, corresponding to about 45% of the available area. Migrating cells treated with 0.06 µM Cytochalasin D covered a 5.5 x 105 µm2 area or about 28% area closure, while 4 µM Cytochalasin D completely prevented 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).

(B) Dose-response curve for Cytochalasin D. Data are presented as the average percent inhibition of SKOV-3 cell migration into the Detection Zone ± SD from 24 replicates. An IC50 of 0.09 µM and a Z’ factor of 0.59 were calculated. Click Image To Enlarge +

(B) Dose-response curve for Cytochalasin D. Data are presented as the average percent inhibition of SKOV-3 cell migration into the Detection Zone ± SD from 24 replicates. An IC50 of 0.09 µM and a Z’ factor of 0.59 were calculated.

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.

Summary

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.