Experimental Procedure
1. Matrigel Preparation
1) The day before the experiment, remove the Matrigel from the freezer and place it in a 4°C refrigerator overnight to thaw. Meanwhile, pre-cool all materials that will be used.
2) Keep the Matrigel on ice throughout the experiment.
3) Open the sterilized packaging of the angiogenesis slides, and remove the slides.
4) Add 10 μL of Matrigel into each well, ensuring the pipette tip is directly above the center of the well to prevent Matrigel from flowing through to the upper well and leaving residue.
2. Gel Formation
1) First, cover the slides and prepare a 10 cm culture dish with damp paper towels inside to create a humidified chamber.
2) Place the slides inside the culture dish and cover it.
3) Put the entire culture dish into a CO2 incubator and leave undisturbed for approximately 30 minutes for gel formation. Meanwhile, prepare the cell suspension.
3. Cell Seeding
1) Prepare a cell suspension at a density of 2×10^5 cells/ml using digested cells, and mix thoroughly.
2) Retrieve the slides where the Matrigel has solidified.
3) Add 50 μL of cell suspension to each well, ensuring the pipette tip remains vertical above the upper well without touching the gel below.
4) Add cell culture medium, cover, and leave undisturbed. After some time, all cells will settle onto the surface of the Matrigel.
4.Image Acquisition
Observe and photograph the cells according to their growth rate, and save the images.
5.Immunofluorescence Staining (Optional)
1) Carefully remove the medium from the wells without disturbing the gel or cellular networks. Dilute Calcein in serum-free medium to a final concentration of 6-8 µg/mL.
2) Add enough staining solution to fully immerse the cells, and incubate at room temperature in the dark for 30-40 minutes.
3) Wash three times with PBS, adding PBS slowly to the upper well to avoid dislodging cells.
4) Observe fluorescence using Ex=485 nm and Em=529 nm wavelengths.
6. Result Quantification
Measure and record tube length, coverage area, number of loops, and nodes, then perform statistical analysis.
Notes
1) All tips, plates, and EP tubes that come into contact with Matrigel must be pre-cooled.
2) Avoid air bubbles during Matrigel spreading as they can interfere with observation.
3) Ensure uniform distribution of cells when seeding to prevent aggregation which may affect even tube formation.
4) Handle gently when seeding cells to avoid disrupting the flatness of the gel, affecting imaging.
5) Maintain the temperature of the cell suspension; lower temperatures can cause the gel to melt, making it difficult for cells to migrate and differentiate into tubes after inoculation.