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: Once the experiment begins, all steps involving the matrix must be performed on ice. Place the pre-chilled 24-well plate, pipette tips, and thawed matrix on an ice bath.
Coating & Polymerization: Using pre-chilled tips, add 250 µL of the matrix solution to each well of the pre-chilled 24-well plate. Gently swirl the plate to ensure even coverage and avoid air bubbles. Transfer the plate to a 37°C incubator and let it stand for 30 minutes to 1 hour to allow the gel to polymerize completely.
2. HUVEC Preparation & Seeding
1)Cell Status: Use Human Umbilical Vein Endothelial Cells (HUVECs) at passage 3 with good growth status, treated when confluence reaches approximately 80%.
2)Digestion: Aspirate the old medium and gently rinse the cells with PBS 2-3 times. Add an appropriate amount of 0.25% Trypsin. Once the cells become rounded, terminate digestion with complete medium and pipette gently to create a single-cell suspension.
3)Collection & Counting: Collect the cell suspension into a centrifuge tube and centrifuge at 800 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of fresh complete medium. Mix 10 µL of cell suspension with 90 µL of medium for dilution and counting.
4)Seeding: Based on the count, prepare the cell suspension using complete medium to a density that allows seeding 1.0×10⁵ cells per well (100,000 cells/well). After the matrix has fully polymerized, carefully aspirate any liquid from the wells. Slowly add the prepared cell suspension onto the surface of the gel. It is recommended to set up at least 3 replicate wells for each condition.
3. Culture, Observation & Phenotype Recording
- Incubation: Place the seeded 24-well plate in a 37°C, 5% CO₂ incubator.
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Dynamic Observation: Starting from seeding, observe under an inverted microscope every 2 hours. Record the time points of key morphological changes, including cell adhesion and spreading, formation of linear connections, and the development of closed mesh-like structures (indicating the maturation of vessel-like lumens).
Figure 1: Culture status at 8 hours
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.