Reagents List
|
Product Name |
Cat.NO. |
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40183ES |
I. Cell Preparation and Suspension Preparation
- Cell Culture and Status: Use human breast cancer cell line MCF7 in the logarithmic growth phase with viability > 95%. For subsequent live imaging, select cell lines stably expressing luciferase or fluorescent proteins.
- Cell Harvesting and Washing: Digest cells with trypsin and terminate digestion with serum-containing medium. Centrifuge the cell suspension and wash the pellet twice with sterile, pre-chilled PBS or HBSS to thoroughly remove serum and trypsin.
- Cell Counting and Resuspension: Accurately count cells using a hemocytometer or cell counter. Calculate the total required cell number based on the target injection dose (1×10⁶ cells/mouse) and the planned number of mice (typically preparing an additional 10-20% extra). Resuspend the cell pellet in pre-chilled serum-free medium, PBS, or HBSS to the target concentration.
II. Preparation of Cell-Matrigel Mixture
- Low-Temperature Operation: All steps must be performed on ice to prevent Matrigel solidification.
- Reagent Preparation: Thaw Ceturegel® Matrigel overnight at 4°C before use and maintain it at low temperature throughout.
- Equal-Volume Mixing: Calculate the required volumes of cell suspension and Matrigel based on the predetermined injection volume (50 µL/mouse) and Matrigel ratio (typically 1:1 volume ratio).
- Mixing Procedure: Slowly add the calculated volume of pre-chilled cell suspension to an equal volume of pre-chilled Matrigel. Gently pipette to mix using a pre-chilled pipette tip, avoiding bubble formation. Immediately place the mixture on ice after mixing and complete injections within 30 minutes.
III. Orthotopic (Mammary Fat Pad) Inoculation in Immunodeficient Mice
- Animal Preparation: Use 4-6-week-old female immunodeficient mice housed in SPF barrier facilities.
- Anesthesia and Fixation: Anesthetize mice using inhaled isoflurane (recommended) or injectable anesthetics, then fix them in a supine position on the operating platform.
- Localization and Skin Preparation:
Localization: Identify the 4th pair (inguinal) mammary fat pad, located near the base of the hind limb.
Skin Preparation and Disinfection: Remove hair from the injection area using depilatory cream or a shaver. Disinfect the skin alternately with alcohol swabs and iodophor three times.
- Injection Procedure:
Sample Aspiration: Gently mix the cell-Matrigel mixture on ice before injection.
Percutaneous Injection: Gently lift the skin beside the 4th pair of nipples with forceps. Insert the needle approximately 2-3 mm lateral to the nipple, advance parallel to the body surface to the center of the mammary fat pad, and inject slowly. A gel-like should be visible subcutaneously.
- Postoperative Care: Transfer mice to a clean cage and place on a heating pad until fully awake.
IV. Experimental Results
After tumor formation, intraperitoneally inject D-Luciferin substrate and detect bioluminescent signals 10-15 minutes later using an imaging system such as IVIS to quantitatively assess tumor burden. Live imaging results show concentrated tumor formation with strong signals, indicating favorable tumor growth.
Figure: Mouse live imaging (Data source: Peking University)
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°C. 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.