Tumour Invasion Assay (TIA) can be applied to (1) study the effects of various cytokines on the invasion and metastasis of malignant tumor cells; (2) research on some new drugs that inhibit angiogenesis; and (3) study the mechanism of tumor cell invasion and metastasis.
Operation method
Matrigel Matrix Membrane Model
Principle
Matrigel, a matrix component derived from mouse EHS sarcoma containing LN, type IV collagen, contact protein and heparin sulfate polysaccharide, is laid on polycarbonate filter membranes free of polyethylene pyridoxine, and can be reconstituted to form a membrane structure in DMEM medium, which is remarkably similar to the structure of natural matrix membranes. This membrane structure is very similar to the natural matrix membrane structure. The pore size of the filter membrane is generally 8um, and the pores of the membrane are covered by Matrigel, so the cells can not pass through it freely, and they have to secrete hydrolyzing enzymes and pass through the Martrigel-covered membrane by deformation movement, which is more similar to the situation in vivo. The Martrigel-lined membrane was placed between the upper and lower chambers of the Blind Well chamber or the MICS chamber, with the Martrigel-lined side facing the upper chamber, and a chemotactic agent was added to the lower chamber, such as a certain concentration of LN, FN, or mouse 3T3 conditioned medium or human testicular epithelial fibroblasts, and the resuspended tumor cells were added to the upper chamber, and the cells with the ability to invade began to move across the membrane under the induction of chemotactic agent. started to move through the membrane. The time taken for the cells to cross the membrane was related to the amount of Martrigel, and it was more appropriate to choose 25ug Martrigell to spread the membrane and observe the results after 16 hours. Most of the cells crossing the membrane were adhered to the lower surface of the membrane, so the cells on the upper surface could be swabbed off, and then the membrane was fixed with ethanol, stained with PE, and observed under the light microscope to count the number of cells crossing the Martrigel. Alternatively, reconstruction of stromal membrane invasion analysis can be performed with a Transwell. In this method, 30ug of Martrigel was spread on the filter membrane of the upper chamber of the hanging basket type of the Transwell, and the results were observed after adding cells for 72h. It is worth noting that after 72 h of cell incubation in the TransWll chamber, a considerable number of cells crossing the filter membrane no longer adhered to the lower surface of the filter membrane, but were shed into the lower chamber solution. Therefore, this part of cells should be taken into account when counting the number of cells crossing the stroma membrane. The ability of tumor cells to cross the reconstructed stromal membrane shows a good correlation with its in vivo invasive metastatic ability, and the reconstructed stromal membrane model can be used to initially screen anti-invasive drugs. In the above analysis, if the 8um pore size filter membrane is directly placed between the upper and lower chambers of the invasion chamber without laying Martrigel on the membrane, the cells cross the filter membrane by deformation movement, and this model is useful for analyzing the cell motility and the effect of drugs on the cell motility. In addition, the effect of drugs on the chemotaxis or consolidation of tumor cells can be analyzed by adding LN or FN in the lower chamber or by spreading LN or FN on the lower surface of the filter membrane.
Materials and Instruments
Matrigel Move I. Preparation . Dissolve the gel, overnight at 4C. . The substrate gel tends to gel at room temperature, so the tubes and tips used in Steps 2 and 3 should be pre-cooled at -20C prior to the test. Encapsulation of basement membrane (on ice) . Dilute Matrigel gel with serum-free cold cell culture medium DMEM. . Take 100 ul of diluted gel and add it to the upper chamber of a 24-well transwell. . Incubate the transwell at 37°C for at least 4-5 h . III. Hydrate the basement membrane . Lightly wash the gel with serum-free medium . IV. Preparation of cell suspension and chambers . Digestion method to obtain cells from cell culture flasks. . Wash 3 times with medium. . Resuspend cells, 5×105cells/ml, 1% FBS . . Add 200 ul cell suspension to the upper chamber. . Add 600 ul of cell culture medium containing 5 ug/ml fibronectin as adherent subfamily in the lower chamber. Incubation Incubate at 37°C for 20 - 24 h. VI. Staining and counting . Cotton swabs were used to wipe away non-invasive cells on top of the upper chamber. . Transwells were removed, inverted, and air-dried. . 500 μl containing 0.1% crystal violet was added to the 24-well plate, and the chambers were placed in it so that the membranes were submerged in the culture medium, and then taken out after 30 min at 37°C and washed by PBS. . fields of vision were taken on the diameter, photographed, and counted. . 24-well plate was added with 500 μl 33% acetic acid, place the chambers in it, soak the membrane, oscillate for 10 min and fully dissolve. Remove the chambers, and measure the OD value of the 24-well plate at 570 nm on an enzyme labeler to indirectly respond to the number of cells. Caveat 1. It must be dried before photographing. When photographing, place the chamber squarely on the slide and observe and photograph it under an inverted microscope. 2. Before using Matrivgel, it should be transferred from -20℃ to 4℃ and allowed to dissolve naturally (e.g., left overnight), avoiding repeated freezing and thawing. 3. tubes, pipette tips, etc. that need to come into contact with Matrivgel during use should be pre-cooled at 4°C. 4. Pay attention to the aseptic operation when using Matrivgel. Common Problems Solution Preparation 1. DMEM (1) NE---A solution (1 μg/μl, 1 mg/ml) (2) mother liquor 0.1 ml (5 mg) + ddH2O up to 5 ml; filtered and sterilized, stored at -20°C. 2. NE-DMEM (-6 M) NE---A solution (1 μg/μl, 1 mg/ml) 20.5 μl DMEM UP TO 100 ml Filter sterilized and stored at 4°C 3. NE+CGRP-DMEM (-6 M, 100 ng) (1) NE - Liquid A (1 μg/μl, 1 mg/ml) 20.5 μl (2) CGRP-storage solution 50 μl (3) DMEM UP TO 100 ml (4) Filter sterilized and stored at 4°C 4. NE+IFN-DMEM (-6 M, 50 ng) (1) NE---A solution (1 μg/μl, 1 mg/ml) 20.5 μl (2) IFN-γ (25 ng/μl) 25 μl (3) DMEM UP TO 100 ml (4) Filtered and sterilized, stored at 4°C. 5. low serum DMEM medium (upper chamber) 6. 20% FBS-DMEM medium (lower chamber) 7. Matrigel matrix gel (1) 10 mg/ml, 5 ml, dispense into 0.5 ml/only 10 EP tubes. (2) Add 0.5 ml of DMEM when used. (3) Matrivgel maintains a liquid state on ice and rapidly sets to a gel at room temperature. For more product details, please visit Aladdin Scientific website.
DMEM Crystalline Violet Dye Solution Acetic Acid Low Serum DMEM Medium FBS-DMEM Medium IFN-γ
24-transwell (Coster) tubes Refrigerator Centrifuge Pipette gun Guns Tips Gun cases
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