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: Prior to the experiment, place the Ceturegel™ Matrix on an ice pack inside a 4°C refrigerator to thaw overnight.
- Cold Chain Operation & Coating: Perform all steps on ice. Using pre-chilled tips, dispense 200 µL of the thawed matrix solution into the center of the bottom of each well in a pre-chilled 24-well plate. Carefully use the tip to spread the gel evenly, taking care not to touch the well walls.
- Pre-treatment & Polymerization: Place the coated 24-well plate in a 4°C refrigerator overnight for low-temperature pre-treatment. The next day, remove the plate and transfer it to a 37°C incubator to solidify for 1 hour, allowing the matrix to fully transform into a solid gel.
2. HUVEC Preparation & Seeding
- Cell Digestion: Harvest healthy HUVEC cells and digest them using trypsin. Terminate digestion with complete medium and gently pipette to form a single-cell suspension.
- Counting & Resuspension: Count the cell suspension precisely and adjust the concentration to 2×10⁵ cells/mL using complete medium.
- Seeding: After the matrix has solidified, carefully aspirate any liquid from the wells. Add 500 µL of the cell suspension to the surface of the gel in each well, resulting in a total of 1.0×10⁵ cells per well (2×10⁵ cells/mL × 0.5 mL). Gently swirl the plate to ensure even cell distribution.
3. Culture, Observation & Phenotype Recording
- Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ constant temperature incubator.
- Dynamic Observation: Observe morphological changes under an inverted microscope (4× or 10× objective recommended) at 2 hours, 4 hours, and 12 hours post-culture. Detailed records should be made of phenotypic stages, including cell adhesion and spreading, initial formation of linear connections, and maturation of the tubular network (where closed ring-like structures may appear).
4. Experimental Results
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.