See the Cell Migration/Invasion Overview for background information. The examples below demonstrate the use of ACEA’s cell invasion and migration plate (CIM-Plate®) and the xCELLigence® RTCA DP system to study chemotactic migration, with specific focus on inflammation and cancer metastasis.
Application Highlight: Real-Time Chemotaxis of Macrophages
Macrophage recruitment to sites of tissue injury, inflammation, or infection is essential for mounting an effective immune response. Chemotactic migration assays are an invaluable tool for studying inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Iqbal and colleagues (PLoS ONE. 2013 Mar; 8(3): e58744) used the xCELLigence RTCA DP system and CIM-Plate to quantify the migration of murine macrophages in response to CC chemokines and other chemoattractant signaling molecules (Figure 1). They stated that “the xCELLigence real-time migration assay offers a rapid and reproducible system to monitor macrophage chemotaxis,” and “Quantification is also far more rapid and accurate compared to the modified Boyden chamber where multiple images are required followed by staining and counting which can also introduce operator bias”.
Figure 1. Real-Time Chemotaxis of Macrophages. (A) Real-time analysis of murine macrophage migration in the presence or absence of the chemoattractant CCL5. In the absence of cells (negative control; blue line) the impedance signal is unchanged over the 75 minutes of the assay. Though some cells do migrate through the porous membrane in the absence of chemoattractant (green line), macrophage migration is significantly stimulated when CCL5 is present in the lower chamber of the CIM-Plate (red line). (B) Scanning electron microscopy images, at two different magnifications, for all three conditions tested. The large, light grey circles are the gold electrodes. The 8 mm pores that pass through both the membrane and the gold electrodes are dark with a more irregular shape. The electrodes and pores are easiest to discern in the sample lacking cells (right column). Consistent with the impedance trace in (A), these microscopy images show that CCL5 stimulates macrophage migration through the pores (at which point the cells adhere to the underside of the membrane and the gold electrodes). (C) Quantitative representation of the imaging data from (B). The number of cells adhered to individual gold electrodes were counted. Figures adapted from PLoS One. 2013 Mar; 8(3): e58744.
Application Highlight: Chemotactic Migration of Breast Cancer Cells
With metastasis posing the primary challenge in the clinical management of breast cancer, there is high demand for effective diagnostic and therapeutic strategies focused on this facet of the disease. Boggs and colleagues (Cancer Res. 2015; 75(1):203-15) identified a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer. Using the xCELLigence RTCA DP system and CIM-Plate they demonstrated that BT-A549 cells migrate less efficiently when expressing an α-tubulin mutant (K40R) that cannot be acetylated (Figure 2A). These data were corroborated by an orthogonal assay in which migrated cells were imaged on the surface of the gold microelectrodes (Figure 2B).
Figure 2. Chemotactic migration of breast tumor cells is stimulated by α-tubulin acetylation. (A) Analysis of BT-549 cell migration in real-time using the xCELLigence RTCA DP system. Cells were plated in serum-free media (upper chamber) and allowed to migrate toward the lower chamber (containing 5% FBS) for 24 hours. α-tubulin Ctrl: BT-549 cell line stably expressing α-tubulin; K40R α-tubulin: BT-549 cell line stably expressing the K40R α-tubulin mutant that cannot be acetylated; serum-free control: the lower chamber contained serum-free media (and therefore lacked chemoattractant). Error bars represent standard deviation. (B) Imaging of migrated cells. At the 24 hour time point cells on the underside of the upper chamber were fixed and stained. The differences in migrated cell numbers visualized here correlate with the quantitative real-time impedance data from panel “A”. Figures adapted from Cancer Res. 2015; 75(1):203-15.
Key Benefits of xCELLigence for Chemotactic Migration Studies:
- Quantitative monitoring of cell migration in real-time.
- Label-free assay requires no fixation, staining or any other sample processing, dramatically reducing hands-on time.
- Easy quantification of the kinetics of cell migration.
- Rapid optimization of cell density and other assay conditions.
- Non-invasive nature of the assay allows for cells on either side of the microporous membrane to be analyzed by complementary techniques (imaging, RT-PCR, etc.)
To see examples of how the xCELLigence RTCA DP system and CIM-Plate have been used for other types of cell migration/invasion studies, click the below links:
- Wound Healing
Featured xCELLigence RTCA System for Cell Migration Studies
|xCELLigence RTCA DP|
Visit the xCELLigence RTCA DP product page for additional information.