Materials:

• Cultured tumor cells (200,000 cells in 40 µL saline per mouse)
• 1.5 mL Eppendorf tubes
• Sterile 27-32 G needles (A Pro tip: Adjust needle size by checking if counted cells stay alive in vitro after drawing them and dispensing in plate vs. the exact number routinely seeded – if not, lower the G of your needle)
• 1 mL syringe
• Isoflurane
• Q-tips (used for iodine solution and eye cream applications; to give an extra stretch to the skin)
• Iodine solution
• Balb/c mice
• An eye cream that prevents dryness
• Gauze Pads

Preparation of Cells:

1. Mix the tumor cell suspension well but gently.
2. Draw the cells without the needle attached.
3. Attach the needle (to remove bubbles) and adjust the tip to align with the scale.
   A Pro tip: If you want to eliminate bubbles, draw more L than you need. You’ll need to draw some air into the syringe to increase the distance between the fluid level and the tip. To remove bubbles, flick the syringe. Empty all air and dead space until you see the first drop at the tip of the needle. Make sure you leave only the volume necessary for injection.

Preparation of Mice:

1. Weigh and number the mice.
2. Anesthetize the mice with isoflurane.
3. Cover the eyes of the mice with protective ointment to prevent dry eyes.
4. Lay the mouse on the gauze pad before shaving.
   A Pro tip: It is easier to collect hair with gauze pad.
5. Shave the mice.
6. Remove all the hair with the gauze pad.
7. With a Q-tip dipped in an iodine solution, scrub the injection area once.
   A Pro tip: Iodine solution stains all the skin except the nipple area, which makes it easier to identify injection sites.

Injection Technique:

1. Identify the fourth abdominal mammary gland.
2. Lift the skin (3-4 mm) near the fourth mammary gland nipple area while placing the forceps (1-2 mm above the nipple) before injecting.
   A Pro tip: Some mice may have stretchy skin that obstructs vision. To improve access, ask your colleague to gently stretch the skin above and below the injection site using Q-tips.
3. Insert the needle (2-3 mm) from the side at an angle of 180° under the nipple area. The tip should be inserted with an additional 0.5-1 mm.
   A Pro tip: Once the needle has passed the skin barrier and been positioned at the correct depth, lift the tip of the needle with the nipple. The cells are injected beneath the nipple.
4. Inject 40 µL (not more than 50 µL) of the tumor cell suspension.
5. Remove the needle slowly to avoid leakage.
6. Injecting properly will form a visible, palpable round bump that disappears without leaking any cells.
7. Observe the animal after waking up from anesthesia in its follow-up cage.
8. Return the animal to its original cage.
   
   Tumor growth and development should be monitored twice a week in the mice. Depending on the cell type and number injected, tumors are palpable approximately seven days after injection and can be measured with calipers. It should take 2-3 weeks for tumors to form a tumor that can be removed easily.
   
   It is highly recommended that you watch this video, even though the injection technique is different, in order to see how the fourth mammary gland nipple area is identified and what the bump looks like.