Angiogenesis Assay Case Study 5

Experiment Products Used

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

The Matrix Thawing: Prior to the experiment, transfer Ceturegel™ Matrix from a -20°C or -80°C freezer to a 4°C refrigerator and allow it to thaw completely overnight.
Equipment Pre-chilling & Sterilization: Prepare a box of pipette tips pre-chilled to 4°C for aspirating the matrix. Before starting the experiment, place the 24-well plate on an ice pack and sterilize under a UV lamp.
Matrix Dilution: According to product instructions, dilute the matrix using DMEM high-glucose medium without antibiotics or FBS.
Coating & Pre-treatment: Place the fully thawed matrix on an ice pack and gently invert a few times to mix. Place the pre-chilled tips and 24-well plate on ice. Using a 200 μL tip, add 10-20 μL of the diluted cold matrix to each well. Gently spread the gel into a circle with the tip, taking care not to touch the well edges to avoid wasting the gel. Add PBS to the gaps between wells to prevent medium evaporation. Place the coated 24-well plate in a clean sealed bag and store in a 4°C refrigerator overnight.
Gel Solidification: After overnight treatment (approx. 12 hours), remove the 24-well plate and transfer it to a 37°C, 5% CO₂ cell culture incubator. Let it stand for 30 minutes to promote gel polymerization and stabilization.

Cell Source & Status: Use healthy HUVEC cells at passages 3-5, reaching approximately 80% confluence 24 hours before the experiment.
Serum Starvation: Replace the complete medium with DMEM containing only 0.2% FBS and culture for 24 hours to synchronize the cell cycle.
Digestion & Resuspension: After starvation, aspirate the supernatant and wash the cells with PBS. Add trypsin solution containing EDTA to digest the cells and detach them. Collect the cell suspension and perform precise counting using a cell counter. Resuspend the cells in DMEM medium containing 10% FBS.
Seeding: Seed the cell suspension onto the pre-solidified gel substrate at a concentration of 1.5×10⁵ cells per well in 500 μL. When seeding, keep the tip vertical and add the suspension slowly to avoid damaging the gel with impact. Gently swirl the plate to ensure even cell distribution.

Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator for continued culture.
Dynamic Observation: Observe HUVEC tube formation using an inverted microscope at 2, 4, 6, and 8 hours post-seeding. Detailed records should be made of the time points for 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.