Angiogenesis Assay Case Study 4

Experiment Products Used

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

Cell Source: The cells used were Passage 5 HUVECs (non-primary), purchased from the Cell Bank of the Chinese Academy of Sciences.
Matrix Thawing & Consumables Pre-chilling: One day prior to the experiment, place the matrix gel in a 4°C refrigerator to thaw overnight. Simultaneously, place the required plates and pipette tips in a -20°C freezer to pre-chill. Turn on the ice maker in advance, fill an iron box with crushed ice, seal with plastic wrap, and UV irradiate for 20 minutes before use.
Cold Chain Operation: All steps involving the matrix gel must be performed on ice using pre-chilled consumables.
Coating & Polymerization: Using a pre-chilled tip, vertically dispense 30 µL of the thawed matrix gel into the center of the bottom of a 24-well plate (do not fully depress the plunger to prevent air bubbles). Use a 1 mL tip to gently spread the gel, ensuring the tip does not touch the well walls. Place the coated 24-well plate in a 4°C refrigerator for 2 hours, then transfer it to a 37°C incubator to solidify for 1 hour, allowing the matrix to fully polymerize.

Cell Pre-treatment (12-well Plate Seeding & Drug Addition): Seed HUVEC cells into a 12-well plate at a density of 1.5×10⁵ cells per well and culture overnight to allow adhesion.
Starvation: Discard the original medium, replace with complete medium containing 2% serum, and add the corresponding drug. Perform starvation treatment for 12 hours (specific time can be adjusted via pilot experiment based on drug toxicity).
Digestion & Counting: After drug treatment, digest the cells, centrifuge to obtain the cell pellet, resuspend in complete medium containing growth factors, and perform precise counting.
Seeding: After the matrix gel in the 24-well plate has fully solidified, carefully aspirate any liquid from the wells. Adjust the cell density and seed 9-12×10⁴ cells (90,000 to 120,000 cells/well) onto the gel surface in 500 µL of complete medium (containing growth factors) per well.
Distribution: After seeding, gently tap the sides of the plate to ensure even cell distribution.

Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator.
Dynamic Observation & Recording: Starting from seeding, observe under an inverted microscope every 1 hour. The optimal time for tube formation varies by experimental conditions. Based on your experience, the best tubular structures form at 3 hours, and the network begins to show slight collapse after 4 hours. Detailed records should be made of morphological changes at key time points, including cell adhesion and spreading, formation of tubular connections, and network 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.