Construction of a subcutaneous hormonal tumor model of melanoma
["Collaborating Expert | Huizhen Lin, M.S.", "Basic Medical Immunology Sun Yat-sen University"], ["Reviewed Expert | Dr. Leyi Zhang", "Clinical Medicine Zhejiang University"]
Summary
The B16F10 melanoma subcutaneous hormonal model is simple to operate, low cost, reproducible, and has a relatively short experimental period, making it a commonly used animal model for melanoma research.
Principle
Mice were given subcutaneous injections of B16F10 tumor cells, which rapidly proliferated and grew to form melanomas.
Appliance
It can be used to study the role of target genes, drugs, etc. in the development of melanoma.
Operation method
Mouse melanoma subcutaneous loaded tumor model
Principle
Mice were given subcutaneous injections of B16F10 tumor cells, which rapidly proliferated and grew to form melanomas.
Materials and Instruments
1. B16F10 tumor cells;
2. C57BL/6 mice;
3. Reagents: PBS buffer, trypsin solution, 75% alcohol disinfectant;
4. equipment: centrifuge, 15 ml centrifuge tube, 1.5 ml EP tube, microscope, 1.5 ml syringe, mouse shaver, sterilized cotton balls, vernier calipers.
Move
1. Digest B16F10 tumor cells in logarithmic growth phase into a single cell suspension using trypsin solution, transfer to a 15 ml tube, adjust the centrifuge speed to 1600 rpm, centrifuge for 5 minutes and discard the supernatant. 2. Resuspend the cells by adding 5 ml of PBS buffer, wash and discard the supernatant;
2. resuspend the cells with 5 ml of PBS buffer, wash, and centrifuge at 1600 rpm for 5 minutes; discard supernatant;
3. Resuspend the cells with 1 ml of PBS buffer and count the cells using a microscope. 4;
4. Add PBS buffer according to the counting result, adjust the cell concentration to 5×106/ml, and transfer the cells to 1.5 ml EP tubes;
5. Control the weight of mice at 18~22 g during inoculation. Use a mouse shaver to remove the hair on the lower right back of the mice, expose the skin, and sterilize it with 75% alcohol disinfectant;
6. Blow and mix the cell suspension again. Draw up the cell suspension with a 1.5 ml syringe, insert the syringe needle with the beveled side facing upward into the subcutaneous area of the mice, and slowly inject 100 ul of cell suspension, so that each mouse will be inoculated with 5×105 cells, and then rotate the needle to exit the needle, and then use a sterilized cotton ball to press on the injection site for 1~2 minutes;
7. closely monitor the growth of the tumor after injection, and when a black hard mass is seen at the tumor site on about the 7th day, the modeling is successful;
8. Use vernier calipers to measure the tumor volume every 1~3 days, and record the longest (a) and shortest (b) diameters of the tumor, and calculate the tumor volume: V = a × b × 0.5 × b ( mm3 ).
Caveat
1. Before injecting B16F10 cells, the cells should be washed 1~2 times with PBS buffer to avoid the serum and other components in the cell culture medium from causing rejection in mice and affecting the growth of tumors in mice;
2. After the injection is completed, the needle should be rotated out and the injection site should be pressed with a sterilized cotton ball for 1~2 minutes to avoid oozing of the cell suspension;
3. B16F10 melanoma grows faster in mice subcutaneously, so we should pay attention to control the experimental period to avoid excessive tumor growth beyond the ethical limits of animals due to the long experimental period.
Common Problems
1. large differences in tumor growth rate between mice: this may be due to the fact that the cell suspension was not adequately mixed before the syringe was drawn up, resulting in inconsistency in the actual number of cells injected into each mouse;
2. no melanoma hard mass is seen on day 7: since the cells in each laboratory come from different sources and have different states, there may be differences in growth characteristics, so the number of cells injected can be adjusted or the observation time can be extended;
3. separation of the tumor reveals a black liquid inside the tumor: B16F10 melanoma is soft in texture, and a large amount of black pasty tissue after incision is a normal phenomenon.
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