Reagents List
|
Product Name |
Cat.NO. |
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40183ES |
I. Cell Preparation and Suspension Preparation
- Cell Culture: Use mouse lung cancer cell line LLC, ensuring cells are in the logarithmic growth phase and in good condition.
- Cell Harvesting and Washing: Digest cells with trypsin, neutralize with serum-containing medium, and centrifuge. Wash the cell pellet 1-2 times with pre-chilled PBS to remove residual serum.
- Cell Counting and Resuspension: Perform accurate cell counting. Based on the experimental design of injecting 2×10⁵ cells per mouse, resuspend cells in pre-chilled PBS to the target concentration.
II. Preparation of Cell-Matrigel Mixture
- Low-Temperature Operation: All operations must be performed on ice to prevent Matrigel solidification.
- Equal-Volume Mixing: Gently mix the prepared cell suspension with pre-chilled Ceturegel® Matrigel at a 1:1 volume ratio in a pre-chilled centrifuge tube, ensuring uniform mixing without bubbles.
- Keep Cold for Use: Immediately place the mixture on ice after mixing and use it for inoculation within 30 minutes to maintain cell viability and Matrigel fluidity.
III. Mouse Orthotopic Inoculation Surgical Procedure
- Animal Anesthesia:
Place the mouse in an anesthesia induction chamber and turn on the anesthesia machine.
Set induction parameters: Adjust isoflurane concentration to 2.5%-3%, oxygen flow to 3 L/min. After approximately 2-3 minutes, the mouse will enter a deep anesthesia state.
Remove the mouse and place it on the operating table, maintaining anesthesia with a mask. Adjust maintenance parameters: Isoflurane concentration to around 1%, oxygen flow 0.5-1 L/min.
Fixation and Disinfection: Fix the mouse in a right lateral position on the operating table. Alternately disinfect the area from the left forelimb axilla to the lower rib margin using 75% ethanol and iodophor.
- Thoracotomy and Orthotopic Injection:
Use sterile surgical instruments to incise the skin and muscle layer along the left rib margin, exposing the 3rd and 4th ribs.
Use a microsyringe (e.g., Hamilton syringe) to aspirate 20 µL of the cell-Matrigel mixture kept on ice.
Between the 3rd and 4th ribs, vertically insert the needle into the lung tissue to a depth of approximately 3 mm.
Slowly and steadily inject the entire suspension.
- Postoperative Treatment:
After injection, slowly withdraw the needle and briefly apply gentle pressure to the puncture site with a sterile cotton swab, observing for any leakage. Use absorbable sutures to close the muscle and skin incisions layer by layer, disinfect the wound again with iodophor, transfer the mouse to a warm, clean cage, monitor closely until fully awake, and observe postoperative status.
IV. Experimental Results
After constructing the orthotopic lung cancer transplantation model, 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 1: Mouse live imaging
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.