Angiogenesis Assay Case Study 7

Experiment Products Used

Product Name	Cat.NO.
Ceturegel™ Matrix (GFR / Phenol Red-Free / LDEV-Free) (Equivalent to Corning Matrigel)	40186ES

Prior to Matrix Thawing: Transfer the matrix stored at –20°C or –80°C to a 4°C refrigerator and allow it to thaw completely (can be left overnight).
Equipment Pre-treatment: Prepare 4°C pre-chilled tips for aspirating the matrix. Before the experiment, place the 24-well plate on an ice pack and sterilize under UV light.
Matrix Dilution: If dilution is required, use DMEM high-glucose medium without antibiotics and FBS according to the instructions.
Coating & Pre-treatment: Place the fully thawed matrix on an ice pack and gently invert to mix. Using pre-chilled tips, add 10–20 μL of matrix to the center of each well in the 24-well plate. Avoid touching the well walls and gently spread it into a circle. Add PBS to the gaps between wells to prevent evaporation. Place the coated plate in a sealed bag and store at 4°C overnight.
Gel Solidification: After 12 hours, remove the plate and place it in a 37°C, 5% CO₂ incubator for 30 minutes to allow the gel to polymerize and stabilize.

Cell Source & Status: Select healthy HUVEC cells at passages 3–5 and treat them when the density reaches approximately 80%.
Serum Starvation: Replace the complete medium with DMEM containing 0.2% FBS and culture for 24 hours to synchronize the cell cycle.
Digestion & Resuspension: After starvation, digest the cells, collect the suspension, and perform precise counting. Resuspend the cells in DMEM containing 10% FBS.
Seeding: Seed the cell suspension onto the pre-solidified gel substrate at a density of 1.5×10⁵ cells per well in 500 μL. Add the suspension slowly and vertically to avoid damaging the gel structure. Gently swirl the plate to ensure even distribution.

Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator for continued culture.
Dynamic Observation: Observe and record tube formation at 2, 4, 6, and 8 hours under an inverted microscope. Detailed records should be made of key morphological changes, including cell adhesion and spreading, formation of linear connections, and tubular network formation.

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.