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
- Consumables Pre-chilling: Prior to the experiment, place Ceturegel™ Matrix, a box of 200 μL tips, EP tubes, and a 24-well plate in a 4°C refrigerator to thaw/pre-chill.
- Cold Chain Operation: Perform all steps involving the matrix on an ice pack.
- Coating & Polymerization: On the ice pack, add 20 μL of the matrix solution to each well of the pre-chilled 24-well plate, avoiding the formation of air bubbles. Use a 1 mL tip to gently spread the gel, then gently swirl the plate to ensure it covers the bottom evenly.
- Humidity Control: Add a ring of PBS around the matrix area to prevent the gel from drying out during incubation. Transfer the coated 24-well plate to a 37°C incubator and let it stand for 0.5-1 hour to allow the matrix to fully solidify.
2. HUVEC Preparation & Seeding
- Drug Treatment: Treat HUVEC cells with the corresponding drugs according to the experimental design.
- Cell Collection & Resuspension: Collect endothelial cells from different treatment groups. Resuspend the cells in phenol red-free DMEM medium containing 10% Fetal Bovine Serum (FBS) and perform precise counting.
- 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, controlling the cell number to approximately 1.0×10⁵ cells per well (100,000 cells/well). Set up 3 replicate wells for each experimental condition to ensure statistical significance.
3. Culture, Observation & Phenotype Recording
- Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator and incubate for 4-8 hours.
- Observation & Imaging: During incubation, observe cell tube formation using an inverted fluorescence microscope and capture images at key time points.
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.