Reagents List
|
Product Name |
Cat.NO. |
|
40183ES |
I. Cell Preparation and Suspension Preparation
- Cell Culture: Use mouse hepatocellular carcinoma cell line Hepa1-6 in the logarithmic growth phase.
- Cell Digestion and Harvesting: Digest cells with trypsin containing 0.25% EDTA, neutralize with serum-containing medium, and centrifuge to collect.
- Washing and Resuspension: Wash the cell pellet twice with pre-chilled PBS, centrifuge, and discard the supernatant. After accurate cell counting, resuspend the cells in pre-chilled PBS and adjust the concentration to 3×10⁷ cells/mL. Keep the suspension on ice.
II. Preparation of Cell-Matrigel Mixture
- Low-Temperature Operation: All operations must be performed on ice to prevent Matrigel solidification.
- Reagent Preparation: Transfer aliquoted Matrigel stored at -20℃ to a 4℃ refrigerator the night before to thaw slowly.
- Equal-Volume Mixing: In a pre-chilled centrifuge tube, gently mix 100 µL of thawed, pre-chilled Matrigel with an equal volume (100 µL) of the above cell suspension (concentration 3×10⁷ cells/mL), avoiding bubbles.
- Keep Cold for Use: Place the mixture on ice immediately after mixing. Immediately aspirate the entire 200 µL of mixture using a pre-chilled syringe for inoculation to minimize Matrigel solidification.
III. Subcutaneous Inoculation in C57BL/6 Mice
Animal Preparation: Restrain the C57BL/6 mouse, remove hair from the left axillary region using a shaver or depilatory cream, and disinfect the skin with 75% alcohol swabs.
Subcutaneous Injection:
- Use a syringe pre-filled with the mixture (typically a 1 mL syringe with an appropriate needle).
- Hold the mouse with your left hand to stretch the skin on the left side. Insert the needle subcutaneously into the disinfected area at a shallow angle (approximately 10-30 degrees) and advance parallel to the body surface to create a subcutaneous tunnel.
- Slowly and steadily inject the entire 200 µL of cell-Matrigel mixture subcutaneously; a round wheal should be visible.
- Wait a few seconds after injection, then slowly withdraw the needle and apply gentle pressure to the puncture site with a dry cotton ball to prevent leakage.
Postoperative Observation: Return the mouse to its cage and observe its short-term status for normalcy.
IV. Experimental Results
Four days after injection, obvious rice-grain-sized tumor nodules can be observed. The figure below shows tumor volume on day 9 post-injection. The tumor volume without Matrigel was the smallest, while the tumor volume in mice using Yeasen Matrigel was the largest.
Figure. Tumor volume comparison across conditions (left–center–right):
No Matrigel (left), ~185.5 mm³; Other brand Matrigel (center), ~188.7 mm³; Yeasen Matrigel (right), ~309.8 mm³ (calculated as 0.5 × 7.7 × 10.45 × 7.7).
Subcutaneous Tumorigenesis Experiment Tips
Cell Status Priority: Ensure the use of cells in the logarithmic growth phase with high viability (>95%). Perform accurate cell counting before inoculation and minimize the time cells spend on ice or in suspension to maintain optimal vitality.
Strict Low-Temperature Operation: Matrigel polymerizes rapidly above 4℃. All steps involving Matrigel (thawing, aliquoting, mixing) must be completed on ice. Syringes should also be pre-chilled; this is key to preventing mixture solidification in the needle and ensuring smooth injection.
Injection Technique: For subcutaneous injection, after the needle pierces the skin, advance it parallel to the body surface for a short distance to form a "tunnel" before slowly injecting the liquid. Gently press the needle site after injection to effectively prevent cell suspension leakage and ensure accurate inoculation volume.
Standardized Monitoring: Once tumors are palpable, it is recommended that the same operator regularly and gently measure them using calipers. Record the longest diameter and the shortest diameter perpendicular to it, and use a uniform formula to calculate volume to reduce human error and obtain reliable growth curves.