IncuCyte® Chemotactic Invasion
Chemotactic cell invasion is the directed movement of cells through an extracellular matrix in response to a chemical stimulus. It is a key component of cancer metastasis where invasive cells of the primary tumor degrade the underlying basement membrane, move toward the circulatory system and disseminate throughout the body. Metastatic potential is associated with tumor cells that have undergone epithelial-mesenchymal transition (EMT) and display a mesenchymal-like phenotype, which includes enhanced migratory capacity and invasiveness.
Cell-based assays enabling real-time visualization and detailed phenotypic analysis of chemotactic cell invasion are critical to further understand the processes involved in cancer metastasis and the discovery of treatments that block metastasis.
A fully integrated solution enabling real-time visualization and automated analysis of chemotactic cell invasion in a 96-well format – all inside your tissue culture incubator.
Directed invasion of HT-1080 fibrosarcoma cells through a 3D matrix of basement membrane extract (BME) toward serum. Visualized using an IncuCyte® ClearView 96-well Cell Migration Plate. HT-1080 cells expressing a nuclear restricted red fluorescent protein (IncuCyte® NucLight Red Essen Bioscience 4476) were visualized and quantified in real time as they invaded through BME gel using the IncuCyte® ZOOM Live Cell Imaging System. Integrated IncuCyte® Chemotaxis Cell Migration software enables easy quantification of cells that have moved through the membrane pores and adhere to the lower surface (indicated as blue objects).
Monitor chemotactic invasion in real time through your choice of 3D extracellular matrix gel. Time-lapse images of a nuclear red labeled HT-1080 tumor cell (black arrow) invading through a matrix of basement membrane extract toward a pore leading to serum (orange circle). The cancer cell extends into and penetrates, the matrix moving toward and through the pore. In the final image, the cancer cell has passed through the pore and has adhered to the underside of the membrane (blue circle) where it is quantified.
Profile inhibitors of tumor cell invasion. IncuCyte® high-definition images of nuclear red labeled HT-1080 fibrosarcoma cells migrating across a 2D substrate and invading through a 3D basement membrane extract (BME) biomatrix in response to a serum gradient – note the differential morphology and invasive filopodia-like projections that extend into the ECM. Time course plots reveal that cell invasion through the BME matrix is slower than migration and that invasion, but not migration, is inhibited by the matrix metalloproteinase inhibitor, GM6001 in a concentration dependent manner.
Cross compare the invasive capacity of cell types in a single plate (e.g. isogenic pairs, tumor biopsies)
Quantify metastatic potential. Comparison of the migration and invasion time-course profiles for a highly invasive (HT-1080 fibrosarcoma) and weakly invasive (NIH-3T3 fibroblast) cell type in the presence of increasing 3D gel density (basement membrane extract). Note that the NIH-3T3 cells are effectively unable to invade through the ECM, but do migrate in the absence of the 3D biomatrix. NIH-3T3 cells show less metastatic potential than HT-1080 cells.
Robust 96-well assays. Representative 96-well microplate graphs showing HT-1080 invasion through a 3D basement membrane extract biomatrix towards serial dilutions of serum to illustrate inter-plate reproducibility. Z’ values ranged from 0.7 to 0.8 for four replicate plates over three days. Corresponding concentration-dependent response curves to FBS provided reproducible measurements of the pro-invasive effects of serum (EC50 value range 1.5% to 3.4%) within and between days.
Membrane-based transwell systems for chemotaxis assays are capable of maintaining chemoattractant gradients for a few hours, but beyond that they collapse, leading to equimolar amounts of chemoattractant on both sides of the membrane. ClearView 96-Well Chemotaxis Plates rely on 96 tiny pores (8 µm in diameter) to set up and maintain the chemoattractant gradient for 72 hours. Since chemotaxis migration assays rely on chemical gradients to differentially induce migratory behavior in cells, maintaining the chemoattractant gradient is essential.
Yes, rather than just citing the reduction of cells on the surface of the membrane, the IncuCyte™ ZOOM can provide real-time analysis of the movement of cells through the pores to the underside of the well. It simultaneously provides live-cell analysis of both surfaces of the insert, enabling morphological analysis of the cells before, during, and after migrating through the pores.
Chemotaxis assays analyze cell migration in response to a chemoattractant, a process that guides cell trafficking in vivo. Examples of chemotaxis assay applications include the analysis of chemotactic migration across substrate surfaces, chemotactic invasion through 3-D biomatrix gels, and chemotactic transendothelial migration of leukocytes (extravasation or diapedesis). Studying chemotaxis requires the generation of chemotactic gradients, as cells travel from areas with one concentration to areas of another (high concentration to low concentration, or low concentration to high concentration). Some chemotaxis assays rely on membranes to maintain these chemoattractant gradients, but those gradients are unstable and they rapidly collapse. ClearView 96-Well Chemotaxis Plates enable gradient maintenance for at least 72 hours -- Something other chemotaxis assay systems can’t claim.
Learn more about the ClearView method for chemotaxis assays
Leukocyte extravasation is the process by which leukocytes in the circulation stop rolling along vascular endothelium and migrate across the endothelium in response to inflammation. In order for any system to accurately recapitulate transendothelial migration, it must first involve the generation of a monolayer of endothelial cells over top of the pore-based insert. Once confluence of the endothelial cells is established, the leukocytes can be carefully added to the culture, where they will extravasate between the endothelial cells in response to the chemoattractant in the reservoir.
The most common effector cells used in the study of trans endothelial migration of leukocytes in vitro include primary T cells, neutrophils, and Jurkat cells. The most commonly used cells to recapitulation the endothelial cell layer are HUVECs. Other cells not listed here that exhibit the ability to extravasate through the endothelium may work with this system, but the protocol – including the choice of endothelial monolayer – will need to be experimentally determined.
While immune cells are non-adherent, their interaction with ECM on the substrate is essential for their migration. Immune cells do not migrate on uncoated surfaces, but can readily migrate in the presence of appropriate ECM proteins. Examples of substrate coatings that are compatible with immune cell chemotaxis include ICAM-1, fibronectin, and Matrigel®/FBS.
An add-on software module for the IncuCyte® instrument. Analyze label-free and fluorescently-labeled chemotactic cell migration images acquired using the ClearView Chemotaxis Plate. This software is required for analyzing chemotaxis using the IncuCyte® instrument.
The IncuCyte® ClearView 96-Well Chemotaxis Plate provides an optically clear surface for label-free imaging and analysis of chemotactic cell migration within the IncuCyte® instrument. The plates are required for analyzing chemotaxis using the IncuCyte® instrument.