Angiogenesis assays model the formation of new blood vessels from existing vasculature, a process essential for wound healing, development, and tumor growth. Matrix Gel provides the structural and biochemical cues that support endothelial cell adhesion, migration, and capillary-like tube formation in vitro, allowing visualization of angiogenic processes in real time.

I. Protocol for Angiogenesis Assay

1. Matrix Gel Preparation

1) The night before the experiment, remove Ceturegel™ Matrix Gel from the freezer and thaw overnight at 4 °C. Pre-chill all labware.

2) Keep Matrix Gel on ice throughout the experiment.

3) Open the sterile packaging of the angiogenesis slide and remove the slide.

4) Add 10 μL of Ceturegel™ Matrix Gel into each well. Hold the pipette tip vertically above the center of the well to prevent Matrix Gel from flowing into the upper chamber and leaving residual gel.

2. Gelation

1) Cover the slide with its lid. Prepare a 10 cm culture dish with wetted paper towels to create a humidified chamber.

2) Place the slide into the dish and cover with the dish lid.

3) Incubate the entire dish in a CO₂ incubator at 37 °C for ~30 min to allow gelation. Meanwhile, prepare the cell suspension.

3. Cell Seeding

1) Prepare a cell suspension at a density of 2 × 10⁵ cells/mL after digestion, and mix thoroughly.

2) Remove the slide with gelled Matrix Gel.

3) Add 50 μL of cell suspension to each well, keeping the pipette tip vertical and avoiding contact with the gel below.

4) Add culture medium, cover the slide, and incubate. Cells will settle onto the surface of the Matrix Gel over time.

4. Imaging

Take photos at regular intervals according to cell growth rate.

5.Immunofluorescence Staining (Optional)

1)Carefully remove the medium without disturbing the gel or cell network. Dilute calcein AM in serum-free medium to a final concentration of 6–8 µg/mL.

2)Add staining solution to fully cover the cells. Incubate at room temperature, protected from light, for 30–40 min.

3)Wash three times with PBS, adding PBS gently to the upper chamber to avoid dislodging cells.

4)Image using Ex = 485 nm, Em = 529 nm.

5. Data Analysis

Measure and record tube length, coverage area, number of loops, and number of branch points, followed by statistical analysis.

II. Experimental Results

Figure 1. Angiogenesis results and immunofluorescence staining images

III.Troubleshooting

Q1: Which cell types are commonly used for angiogenesis assays?

A1: Human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMVECs) are standard choices.

Q2: How long does tube formation typically take?

A2: Tubule structures usually form within 4–8 hours after cell seeding on Matrix Gel.

Q3: Can growth factors be added to enhance angiogenesis?

A3: Yes, supplementing with VEGF, FGF, or EGF can significantly enhance tube formation.

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