Matrigel is a matrix component extracted from mouse EHS sarcoma, containing LN, type IV collagen, contact protein and heparin sulfate polysaccharide, which is spread on a polycarbonate filter without polyvinylpyrrolidone. It can reconstruct a membrane structure in DMEM culture medium, which is very similar to the natural matrix membrane structure. The pore size of the filter membrane is generally 8μm, and the membrane pores are covered with Matrigel. Cells cannot pass through freely, and must secrete hydrolases and deform to pass through the filter membrane covered with Matrigel, which is similar to the in vivo situation. This model can be used to detect cell invasion. The in vitro model is used to detect cell invasion, which is mainly used in the effects of various cytokines on the invasion and metastasis of malignant tumor cells and the study of some new drugs that inhibit angiogenesis.
Experimental steps:
1. Matrigel melts overnight at 4℃
2. Dilute matrigel to a final concentration of 1mg/ml
3. Add the diluted matrigel vertically to the center of the bottom of the upper chamber of the chamber. Incubate at 37℃ for 4-5 h
4. All cell culture reagents and Transwell chambers are placed in a 37℃ constant temperature water bath for incubation
5. Digest the cells cultured to the logarithmic phase, suspend the cells with serum-free medium, count them, and adjust the concentration to 5*105/ml
6. Add medium containing 20% serum to the lower chamber. Add cell suspension to the upper chamber and continue to culture for 24 h at 37 ℃, 5% CO2, and saturated humidity
7. Take out the chamber, dry the liquid in the upper chamber, move it to the wells pre-added with methanol, and fix it at room temperature for 30 min
8. Take out the chamber, dry the fixative in the upper chamber, move it to the wells pre-added with crystal violet stain, and stain it at room temperature for 15-30 min; rinse and soak it with PBS several times, take out the chamber, first use a dry cotton swab to absorb the liquid in the upper chamber, and then use the cotton swab to gently wipe the cells on the surface of Matrigel gel and polycarbonate membrane
9. Peel off the cells on the membrane surface, dry it, and seal the slide
10. Count under a microscope and statistically calculate the results; (If the bottom membrane does not need to be preserved, step 9 can be omitted)
Result example:
