Angiogenesis Assay Case Study 10

Experiment Products Used

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

Matrix Thawing & Consumables Pre-chilling: In advance, take Ceturegel™ Matrix out of the –20°C freezer, place it on ice, and put it in a 4°C refrigerator to thaw overnight. Simultaneously, pre-chill the 24-well plate, 10 μL and 200 μL tips, and ice box at –20°C.
Cold Chain Operation: Perform all steps involving the matrix on ice. Place the fully thawed matrix on ice and gently invert to mix.
Matrix Dilution & Coating: Using pre-chilled tips, dilute the matrix with DMEM basal medium at a 1:1 volume ratio and mix thoroughly. In the pre-chilled 24-well plate, add PBS to the gaps between wells to prevent the gel from cracking during subsequent incubation. Vertically dispense 20 μL of the diluted matrix solution into the center of each well. Use a 200 μL tip to gently push the gel drop to distribute it evenly (no need to spread it too wide, as it will level naturally overnight at 4°C).
Pre-treatment & Solidification: Place the coated 24-well plate in a sealed bag, lay it flat in a 4°C refrigerator overnight to allow the gel to level naturally. The next day, remove the plate and transfer it to a 37°C incubator for 30 minutes to allow the matrix to solidify completely.

Cell Status: Use HUVEC cells at passages 3-5, cultured to approximately 80% confluence within 24 hours.
Serum Starvation: Replace the complete medium with DMEM containing 0.2% FBS and continue culturing for 24 hours to synchronize the cell cycle.
Drug Pre-treatment: After starvation, pre-treat the cells with drugs according to the experimental design for 6-8 hours.
Digestion & Resuspension: After drug treatment, digest the cells and collect them by centrifugation. Resuspend the cells in complete medium containing the corresponding drugs and perform precise counting. Adjust the cell concentration to 2.0×10⁵ cells/mL (200,000 cells/mL).
Seeding: After the matrix has fully solidified, carefully aspirate any liquid from the wells. Add 500 μL of cell suspension to the surface of the gel in each well, seeding 1.0×10⁵ cells (100,000 cells/well). Gently swirl the plate to ensure even cell distribution.

Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂, 90% humidity incubator.
Dynamic Observation: Observe and capture images using an inverted microscope at 0, 2, 4, 6, 8, 9, and 12 hours post-seeding. The optimal time for tube formation can be determined via pilot experiments; generally, vascular networks form within 3-12 hours, and the timing is closely related to cell health. Detailed records should be made of key morphological changes, including cell adhesion and spreading, tubular network formation, and maturation.

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.