Like wild animals, cells migrate from one place to another. Although the cause of cell migration is not clear, it helps us understand the processes of normal cell development, development, differentiation, wound healing, and certain diseases, such as cancer metastasis. A special case of cell invasion is the three-dimensional migration of cells. At the cellular level, cell adhesion plaques (integrin-mediated attachment points of cell motility) often occur during cell migration and invasion. By using sticky spots as a towing point, the cells pull themselves toward their destination. In order to study the complex signals that control these processes, various cell migration detection methods have been developed, such as measuring the number of cells moving from the starting position to the ending position (which may also be dynamic measurements). The four major cell migration experiments were: transmembrane/Boarden chamber, cell scratch assay, cell isolation migration assay (also known as two-dimensional cell migration assay or gap closure assay), and microfluidic technology. Choosing which test to end requires a certain understanding of these tests and what data you need. However, the following problems will be beneficial to the choice of cell migration detection methods.
When should I use a transmembrane/Boyden chamber?
The transmembrane/Boyden chamber is the most commonly used method for cell migration detection. The method began in the 1960s and was named after SVBoyden. The chamber was separated into two by a microporous membrane (usually coated with extracellular matrix proteins). Cavity. Usually, a polycarbonate cell culture nest is placed in the cell culture plate, and the bottom of the nest is a filter. The cells are placed in one chamber (or planted on one side of the membrane) during the assay and a chemotactic agent (or inhibitor) is added to the other chamber. Affected by a chemotactic agent, cells migrate through the pores in the membrane, and then you can use a microscope to observe cell migration. The advantage of this type of experiment is that you can use adherent or suspended cells and can test for migration under chemotaxis gradients. The disadvantage of this method is that the concentration gradient of the chemotactic agent is unknown and variable, and the method can only be used for end point observation, and it is difficult to see the dynamic change process (although this situation is improving, it will be mentioned below). At the same time, manual counting after cell migration is quite tedious, and various migration cell labeling technologies are also being developed. According to Cell Biolabs, "We are very cautious in performing cell labeling, suggesting that cell migration properties after labeling may change, resulting in erroneous results." Cell Biolabs' CytoSelect Cell MigrationAssays Cellular automated technology can be implemented, but cell labeling technology is not used, but techniques are performed using fluorescent dyes after cell migration is completed.
BD Biosciences offers another automated counting solution that uses a special membrane to separate the chambers. The company's FluoroBlok Cell Culture Inserts contain a "light-tight" membrane structure that blocks light penetration of 490-700nn. As a result, researchers can detect fluorescence in the bottom chamber without the light reaching the top of the chamber. With the BD FluoroBlok system, if fluorescently labeled cells pass through the filter membrane, they can be automatically read by the bottom fluorescence reading plate system or fluorescence microscope. This method can be used to dynamically observe cell migration status, not just for endpoint analysis.
BDBioSciences also offers a variety of applications using its FluoroBlok technology. For example, BD BioCoat? Angiogenesis System: Endothelial Cell Migration, which is coated with human fibronectin, is used to detect endothelial cell migration. The BD FluoroBlok Insert system is frequently used for tumor cell, endothelial cell migration and invasion assays.
When should I use cell scratch experiments?
Cell scratch test, also known as wound healing test, refers to the cultivation of cells on a culture dish or plate. After the cells are fused, a line is drawn in the central region. The cells in this line are removed by mechanical force, and then the cells are continued. The culture is observed, and the migration of the cells to the cell-free scratched area is observed to determine the migration ability of the cells. Recently, companies such as Essen BioScience have provided automated cell scratching tools to improve repeatability. The company's CellPlayer Migration Assay (Essen's CellPlayer? Migration Assay) is a 96-well plate scratch detection system. The advantage of this plate is that it is easy to use and can record cell migration in real time. However, since different laboratories may use different scratch test methods, and it is difficult to accurately control the scratched area even with automatic equipment, the scratches of the experimental group and the control group may be identical.
Scratches can also damage the extracellular matrix under the cells. EMD Millipore pointed out that scratch testing is very useful in studying cytoskeletal structure and cell polarity, but because of the uniformity of the instruments used, there are many variables. For example, the size and width of the scratches are inconsistent, which affects the repeatability and consistency of the experimental results. Moreover, cells at the edges of the scratch may be damaged, resulting in difficulty in migration. On the other hand, scratch tests were not able to perform gradient studies of soluble factors and chemotactic agents. Damaged cells may release certain substances into the medium, affecting other cells.
When should I use cell isolation migration experiments?
The cell isolation migration experiment is similar to the scratch test in that the cell migrates into the cell-free region, but the "wound" (cell scratch) in the scratch test does not occur. The cells are grown at the bottom of the culture well, and a substance surrounding the growth of the cells in a certain area is placed around it, for example, a small stopper is placed in the middle of the culture well to produce a cell-free area. At the beginning of the experiment, the small plug was removed and the researchers were able to observe the migration of cells to the cell-free area. The advantage of this method is that cell movement can be observed continuously without cell damage, and the researchers can add some matrix to observe the three-dimensional migration of cells. Disadvantages include the inability to observe suspended cells or chemotactic gradients (measured by the Boyden chamber).
Platypus Technologies and Cell Biolabs use multiwell plates for cell isolation migration assays. Platypus recently introduced a high-throughput compatibility detection solution that can read data through a microscope or high-content imaging instrument. Platypus noted, "Our recently launched ORIS Pro 384-well cell migration assay kit is fully automated, both for tissue culture-treated surfaces and for type I collagen-treated surfaces. ORIS Pro 384 is for 96-well and small plugs The improved detection method, combined with its highly automated configuration, is especially suitable for high-throughput screening. ORIS Pro 384 places a biocompatible gel on the bottom of the culture well without the need to place small plugs in advance or use drugs or Manual removal of small plugs. With the arrival of high-content analytical instruments and multi-parameter reading instruments, researchers can observe the phenotype of cells."
When do you need microfluidic testing?
Cell migration assays based on microfluidic devices are also becoming increasingly popular. The difference between devices is their construction, but generally microfluidic devices contain a channel connecting the ends. Researchers can add at one end and cells can grow at the bottom. The chemotactic agent was then added on the other side, and the cell migration condition was observed using a microscope. The advantage of microfluidic testing is that researchers can save valuable reagents and cells (expensive drugs or rare primary cells), and researchers can also produce linear concentration gradients (as opposed to Boyden chambers). The disadvantage is that due to the small volume, frequent fluid changes may be required to maintain cell viability.
EMDMillipore recently announced a microfluid migration assay kit that can be used alone or in conjunction with the company's Boyden Chamber QCMTM migration and invasive assay kit. “Our company's recently launched Millicell μ-migration Assay Kit, which allows real-time observation of single cell migration. It measures parameters such as cell movement speed, direction and migration index, and supports stable linear concentration gradients, duration More than 48 hours. Therefore, the researchers can distinguish between slow and rapid migration of cells under chemotaxis and random motion. The kit can also use fluorescently labeled cells."
“In June, EMD Millipore will announce the QCM Invadopodia test kit, which contains the cancerous substance invadopodia, which triggers tumor cell migration. It is the first and only reagent to visually quantify the degradation of ECM by various molecules. The kit forms a thin film on the glass culture surface. The compatibilizing reagent co-localizes the actin, nucleus and invadopodial degeneration sites. It can be used to detect the activity of invadopodia inhibitors and enhancers, thus analyzing various Cell type invasive ability. The kit is available in red and green colors.
Platypus pointed out that "Since many researchers are studying anti-cancer mechanisms, cell migration assays should also be closer to physiological conditions. Three-dimensional cell migration assays may be closer to physiological conditions. At the same time, cells may be produced on different culture surfaces. Different signals, the cell surface may be partially in contact with the substrate, and the other part is in contact with the culture medium. Our cell migration products are more inclined to embed all parts of the cell in the matrix. We also need to constantly research to develop into more More detection models."
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