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
- Material Preparation: Gather all necessary materials, including Ceturegel® Matrix, 24-well plates, HUVEC cells, and pre-chilled tips and EP tubes.
- Cold Chain Operation: Before the experiment, place the Ceturegel® Matrix on an ice pack inside a 4°C refrigerator to thaw overnight. Keep the thawed matrix, pre-chilled 24-well plate, and tips on ice throughout the procedure to delay gelation.
- Coating & Polymerization: Using pre-chilled tips, add 50 µL of the matrix solution to the center of each well in the 24-well plate. Gently swirl the plate horizontally on ice to allow the gel to cover the bottom naturally, avoiding air bubbles. Transfer the plate to a 37°C incubator and let it stand for 30–45 minutes to allow the gel to polymerize completely.
2. HUVEC Preparation, High-Glucose Modeling & Seeding
- High-Glucose Injury Modeling: Once HUVEC cells have adhered, initiate the high-glucose injury model.
- Treatment Group: Pre-treat cells for 48 hours using DMEM (low glucose type) supplemented with additional glucose to a final concentration of 6 g/L.
- Control Group: Culture cells using standard DMEM (low glucose type).
- Digestion & Resuspension: After modeling, digest cells from both groups and centrifuge to obtain cell pellets. Resuspend the cells in their respective media (high-glucose or control) and perform precise counting.
- Seeding: Adjust the cell density and seed 1.5×10⁵ cells (150,000 cells/well) in 500 µL of medium onto the polymerized matrix. This density ensures a high probability of cell contact during the initial phase of tube formation. After seeding, gently shake the plate in a "cross" pattern (front-to-back and left-to-right) to ensure even cell distribution.
3. Culture, Observation & Phenotype Recording
Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator.
Dynamic Observation & Recording: Begin observing under an inverted microscope starting at 3 hours post-seeding. Distinct tubular structures typically form around the 4-hour mark. Systematically capture images at this key 4-hour time point. Compare and describe the differences between the high-glucose and control groups regarding tube formation speed, network complexity, and integrity.
4. Experimental Results
Conclusion: Comparative analysis verified the conclusion that "high glucose impairs the formation of tubular structures in endothelial cells."
Figure 1. Control Group (left) & Treatment Group (Right）
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.