Medicilon’s pharmacology services provide you with experiments on tumor formation in nude mice and subcutaneous tumor formation in nude mice. For details, please contact us. Email: marketing@medicilon.com.cn Tel: 021 58591500.
Nude mouse service process
Operation plan design
Method development
Cell preparation
Tumor formation in nude mice
Experimental method of subcutaneous tumor-forming animals in nude mice
Grouping: SW620 cell group, SW620 transfection irrelevant siRNA group, SW620 transfection target gene (6 per group).
Cell preparation and injection of nude mice: Go to the animal room to prepare the experiment.
The untreated mice and the treated mice were taken out of the rat cage and placed in a super clean table in order. The larger mice were weighed, and the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 3 μl/g. After about 5 minutes, the mice began to enter a quiet state, fixing them on their backs. Avoid excessive use of anesthetics.
(1) Spread white cloth on the mouse board, fix the four paws of the mouse in the supine position, and disinfect the ventral side of the abdominal side to the neck, down to the groin, and then to the posterior axillary line. Disinfect twice. Put a surgical towel on the mouse, cut the left side (4*4cm), expose its left abdomen, and disinfect it once with iodophor. Cut the skin 2 cm down at the lower edge of the left costal arch 1 cm to expose the abdominal wall muscles. Pull the abdominal wall muscles with tweezers to avoid the spleen in the abdominal cavity. Cut the abdominal wall muscles to expose the internal organs. Dip the PBS with a cotton swab, set aside the lower end of the spleen, and avoid pulling, so as not to damage the spleen and pancreas.
(2) While finding the spleen, mix 25 μl of Matrigel in the cell tube, suspend the cell pellet, and disperse the cells into a single cell suspension. Aspirate the single-cell suspension in the EP tube with a BD needle, slightly retract the needle core, break out the air bubble, insert the needle into the lower inner side of the spleen (concave surface) and start the injection of cells about 2 mm in the direction of the splenic hilum, and stay for about 30 seconds after the injection. Slowly withdraw the needle to prevent cell leakage. At the same time, use a cotton ball dipped in PBS to compress the surrounding bleeding points to stop bleeding.
(3) Draw about 400 microliters of gentamicin injection (diluted with physiological saline 250:1) with a syringe, inject it into the abdominal cavity, and dip the cotton ball with antibiotics exuded from the margin of the skin. Start suture of abdominal muscles (continuous suture of 4 stitches). Draw about 400 microliters of gentamicin injection into the syringe again to rinse the suture port, and dip the cotton ball to dry. Suture the skin (intermittent suture 4 stitches). Sterilize the suture with an iodophor cotton ball. Remove the fixation so that the mouse is in its natural position and placed in the rat cage.
(4) After all the cells are injected, mark the ear cut, record the ear cut mark and grouping.
Day5-7: Pay attention to the state of the mice and find out if the mice have died due to surgical stimulation, pancreatic injury, infection, etc.
Day42: The cervical spine was dissected, the mice were sacrificed, and the cadavers were dissected to observe the growth status of the spleen tumors, and whether the liver, lungs, and other organs had metastatic tumors. All suspicious tissues are marked and photographed. If there are nodules on the surface, after counting, they are frozen in liquid nitrogen. According to the results, fix it with 10% formaldehyde solution overnight for pathology.
Experimental techniques of subcutaneous tumor formation in nude mice
The state of the cell is the key to the tumor formation experiment, so the growth state of the cell must be good. The cells in the logarithmic growth phase should be taken, and the density of the cells should be about 80-90%; the fresh medium should be replaced the night before the cells are collected.
After digesting the cells with trypsin, wash twice with pre-chilled PBS for the purpose of removing serum from the cells.
Pipette the cell pellet with PBS or serum-free medium to an appropriate concentration. Generally, the amount of cells inoculated for subcutaneous tumors is 1-5×10^6 cells/branch, and the inoculation volume is 0.1 ml, so the concentration of the cell suspension is 1-5×10^7 cells/ml.
After the cells are digested, they should be inoculated subcutaneously to nude mice as soon as possible. Generally, try to complete them within half an hour. Place the cell suspension on ice on the way to reduce the metabolism of the cells.
The selected nude mice are generally 5-8 weeks old, weighing about 18-20g, and choose the areas with abundant blood supply at the planting site, such as the middle and rear of the axilla and the middle and upper part of the groin.
Before the inoculation, blow off the cell suspension with a gun to prevent cell clumping and reduce cell survival rate.
During inoculation, the needle is inserted into the needle a little deeper, about 1cm deep, to reduce the overflow of the cell suspension from the needle eye after injection.
How to grasp the nude mouse? The left thumb and index finger squeeze the skin of the neck of the nude mouse, and the little finger clamps the tail of the nude mouse.
Nude mice tumor formation experiment
A lump can begin to appear under the skin about a week after the inoculation.
According to the characteristics of tumor cells and the amount of inoculated cells, the general period of subcutaneous tumor formation is 1-2 months. The tumor should not grow too large, generally not more than 1000mm3. Excessive tumor load is often made difficult by the magazine for violating animal ethics (because this problem was tragically rejected by plos one).
The measurement of tumor size is generally twice a week, and the vernier caliper measures the longest and shortest parts of the tumor. V=1/2ab2 (a is the long axis and b is the short axis).
After removing the subcutaneous tumor, a part of the frozen nitrogen is used as a protein and RNA for later extraction, and a part of formalin is fixed for immunohistochemistry and immunofluorescence.
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