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 & Consumables Pre-chilling: In advance, take Ceturegel™ Matrix out of the –20°C freezer and place it in a 4°C refrigerator to melt overnight. Place the 96-well plate and sterile pipette tips in a –20°C freezer 2 hours prior to the experiment to pre-chill.
- Cold Chain Operation: Spray the pre-chilled 96-well plate and pipette tips with alcohol for disinfection, then place them in an ice box inside the laminar flow hood.
- Coating & Polymerization: Aspirate the ice-bathed matrix and add it to the 96-well plate at a volume of 45 μL per well. Use the tip to evenly cover the bottom of the well, avoiding contact with the walls. Place the coated plate in a 37°C, 5% CO₂ incubator for 30 minutes to allow the matrix to fully gel.
2. HUVEC Preparation & Seeding
- Cell Status: Harvest endothelial cells during the logarithmic growth phase when confluence reaches 80%-90%.
- Digestion & Resuspension: Wash with PBS 3 times and digest with trypsin. Add an equal volume of complete medium to stop digestion and gently pipette to form a single-cell suspension. Centrifuge at 1000 rpm for 3 minutes and discard the supernatant. Resuspend the cells in pre-warmed medium and perform precise counting. Adjust the cell concentration to 2×10⁵ ~ 5×10⁵ cells/mL and keep the suspension at room temperature.
- Seeding: After the matrix has fully solidified, remove the 96-well plate from the incubator. Add 100 μL of the prepared endothelial cell suspension per well.
- Handling: When adding the sample, touch the tip to the well wall and drip slowly to avoid damaging the gel layer with direct impact. Gently tap the edges of the 96-well plate to distribute the suspension evenly; do not shake vigorously.
3. Culture, Observation & Phenotype Recording
- Incubation: Place the seeded 96-well plate in a 37°C, 5% CO₂ incubator and let it stand.
- Dynamic Observation: Depending on experimental requirements, observe cell tube formation using an inverted microscope at appropriate time points (e.g., 4-8 hours) and record key time points for tubular structure formation.
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.