Angiogenesis Assay Case Study 8

Experiment Products Used

Product Name

Cat.NO.

Ceturegel™ Matrix (GFR / Phenol Red-Free / LDEV-Free) (Equivalent to Corning Matrigel)

40186ES

1. Pre-experiment Preparation & Matrix Coating

  • Matrix Thawing & Consumables Pre-chilling: Take Ceturegel™ Matrix out of the –20°C freezer and place it in a 4°C refrigerator the night before to thaw completely. Simultaneously, pre-chill 200 μL tips at –20°C.
  • Cold Chain Operation: Place the fully thawed matrix on an ice pack and gently invert to mix thoroughly.
  • Coating & Pre-treatment: Using pre-chilled tips, dispense 40 μL of matrix into the center of each well in a 24-well plate. Use the tip to spread the gel in a circular motion, ensuring it does not touch the side walls. Place the coated plate in a 4°C refrigerator overnight to allow it to level naturally.
  • Gel Solidification: The next day, remove the plate and transfer it to a 37°C incubator for 1 hour to allow the matrix to solidify completely.

2. HUVEC Preparation & Seeding

  • Cell Digestion & Counting: Harvest healthy HUVEC cells, perform digestion and centrifugation, and then count the cells precisely.
  • Seeding: After the matrix has fully solidified, carefully aspirate any liquid from the wells. Adjust the cell suspension concentration to seed 2.5×10⁵ cells (250,000 cells/well) per well. Gently add the cell suspension onto the surface of the gel. Swirl the plate gently to ensure even cell distribution.

3. Culture, Observation & Phenotype Recording

  • Incubation: Place the seeded 24-well plate in a 37°C incubator.
  • Dynamic Observation: At 3, 6, and 12 hours post-culture, observe and capture images using an inverted microscope (10× objective). Detailed records should be made of cell adhesion and spreading, tubular network formation, and maturation at different time points.

4. Experimental Results

Figure 1: Culture status at 6h

Figure 1: Culture status at 6h

Angiogenesis Assay Tips

  • Cell Status: Cells at passages 3-5 are recommended; serum starvation can be used for synchronization.
  • Matrix: Keep on ice throughout the process; coat gently and evenly.
  • Seeding: Use a single-cell suspension, count precisely, and add gently.
  • Observation: It is advisable to determine the optimal observation time point via a pilot experiment to capture the network maturation phase.

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