This experimental case is based on feedback from Our Customer.
Protocol Overview
[Note]: *This protocol uses HUVEC cells. Use standard cell passaging procedures with cells at passages 3-5 in good condition, achieving ~80% confluence within 24 hours. Starve the HUVEC cells by replacing the complete medium with DMEM containing 0.2% FBS for 24 hours.*
1. Basement Membrane Matrix Preparation
1) The day before the experiment, take the matrix from the freezer and thaw it overnight in a 4°C refrigerator. Pre-cool all consumables that will contact the matrix.
2) Keep the matrix on an ice bath throughout the experiment.
3) Open the sterile packaging of the angiogenesis slide and remove the slide.
4) Add 10 µL of matrix to each well. Ensure the pipette tip is directly above the well to prevent matrix from flowing into the upper chamber and leaving residue.
2. Gelation
1) First, place the cover on the angiogenesis slide. Prepare a 10 cm culture dish with moistened paper towels to create a humidified chamber.
2) Place the slide into the culture dish and close the lid.
3) Place the entire dish into a CO₂ incubator and let it sit for approximately 30 minutes for the gel to polymerize. Prepare the cell suspension during this time.
3. Cell Seeding
1) After trypsinization, prepare a cell suspension at a density of 2 x 10⁵ cells/mL. Mix thoroughly.
2) Take out the angiogenesis slide containing the polymerized gel.
3) Add 50 µL of cell suspension to each well. Keep the pipette tip vertical above the upper chamber, avoiding contact with the gel in the lower chamber.
4) Add cell culture medium, cover the slide, and let it sit. After a period, all cells will settle onto the surface of the matrix gel.
4. Image Acquisition
Capture images at specific time points, typically at 2, 4, 6, and 8 hours. Tubule network formation is usually visible within 3-12 hours, depending on cell condition.
5. Immunofluorescence Staining (Optional)
1) Carefully remove the medium from the wells without disturbing the gel or cellular networks. Dilute Calcein-AM in serum-free medium to a final concentration of 6-8 µg/mL.
2) Add the staining solution to completely submerge the cells. Incubate at room temperature, protected from light, for 30-40 minutes.
3) Wash three times with PBS. Add PBS gently to the upper chamber to avoid dislodging the cells.
4) Observe fluorescence using Ex=485 nm / Em=529 nm wavelengths.
6. Result Quantification:
Measure and record tubule length, coverage area, number of loops, and branching points. Perform statistical analysis.
Results
Customer Feedback: Tube formation was observed in the 6-8 hour imaging results, with clear and distinct cell clustering. The outcome is highly satisfactory.
Frequently Asked Questions
1. What should I do if the matrix gels too quickly?
Pre-cool all tips, plates, and tubes that will contact the matrix by placing them at -20°C. Perform all matrix handling steps on ice or in a cold environment.
2. What should I do if bubbles appear when plating the matrix?
When plating the matrix into 96-well plates or angiogenesis slides, pipette the liquid carefully and vertically into the wells to avoid bubbles. If bubbles form, centrifuge the plate at 300 x g for 10 minutes at 4°C.
3. During which time window should I observe the cells?
The duration of tubule formation depends on the cell type and extracellular matrix used. It should be determined individually. Typically, HUVECs form tubes within 2-4 hours. After 24 hours, apoptosis begins, leading to detachment from the matrix and tube disintegration.
4. Should I use a gel with or without phenol red in the tubule formation assay?
If using phase-contrast microscopy, phenol red does not interfere and makes handling easier due to its color. However, for fluorescence microscopy, phenol red might interfere with probe wavelengths. In this case, it's better to use phenol red-free gel.
5. Which type of matrix is used for the tubule formation assay?
In principle, any gel can be used for the angiogenesis tubule formation assay. It is important that the cells can attach to the surface. In our experiment, we use a Low Growth Factor Basement Membrane Matrix (Yeasen Cat#40186).
6. What are suitable positive and negative controls for the tubule formation assay?
A positive control is a sample where cells are expected to form tubes, indicating the assay was performed correctly. A negative control is treated identically but is not expected to show any experimental outcome.
7. How do I set up positive and negative controls for the tubule formation assay?
1) For analyzing pro-angiogenic compounds: Use samples treated with known inducers (e.g., VEGF or FGF2) as positive controls.
2) When using primary cells: Pre-screened endothelial cell lines (e.g., HUVEC) showing a defined response to specific growth factors can serve as positive controls.
3) For testing pro-angiogenic substances: Use starvation medium because most cell culture media contain growth factors. To analyze the true effect, both the matrix and medium must be free of growth factors. As a negative control, seed cells on a different matrix (e.g., Collagen I) where tube formation is not expected.
4) For testing anti-angiogenic substances: Use tube formation inhibitors that do not affect cell viability (e.g., Suramin or Sulforaphane) as negative controls.
8. What if I don't have an angiogenesis slide?
Angiogenesis slides provide better imaging, but you can use a 96-well plate as an alternative:
1) Thaw the matrix at 4°C until liquid. Use pre-cooled pipettes or tips to mix the matrix until homogeneous.
2) Keep the fully thawed matrix on ice, invert several times to mix, then add 50-80 µL per well to a pre-cooled 96-well plate.
3) Transfer the plate to a cell culture incubator and incubate at 37°C for 30 minutes for gel formation.
4) Prepare serial dilutions of sample working solutions, setting up test groups, positive control groups, and blank control groups.
5) Digest HUVEC cells from a T75 flask at 80% confluence, centrifuge, and count.
6) Resuspend cells in medium containing 10% FBS to create a single-cell suspension at a concentration of 3-5 x 10⁵ cells/mL.
7) Add 100 µL of cell suspension (containing 30,000-50,000 cells) per well to the 96-well plate.
8) Place the plate in a 37°C, 5% CO₂, 90% humidity incubator. Capture images at 2, 4, 6, and 8 hours. Tubule network formation is usually visible within 3-12 hours.
9) Remaining steps are the same as those described for the angiogenesis slide.
Ordering Information
|
Category |
Name |
Cat. No. |
Size |
Note |
|
Basic concentration of Matrigel 8-12 mg/mL |
40183ES08/10 |
5 mL / 10 mL |
Suitable for 2D/3D culture, invasion and migration assays, and in vivo tumor formation studies. |
|
|
40184ES08/10 |
5 mL / 10 mL |
|||
|
High concentration 18-20 mg/mL |
40187ES08/10 |
5 mL / 10 mL |
High-concentration gel is viscous and gels quickly, ideal for in vivo tumor formation with hard-to-grow cell lines. |
|
|
Ceturegel™ Matrix High Concentration，GFR，LDEV-Free |
40189ES08/10 |
5 mL / 10 mL |
||
|
Ceturegel™ Matrix High Concentration，Phenol Red-Free，LDEV-Fre |
40188ES08/10 |
5 mL / 10 mL |
||
|
Low Growth Factor |
40185ES08/10 |
5 mL / 10 mL |
Minimizes growth factor interference in signaling pathway studies. |
|
|
40186ES08/10 |
5 mL / 10 mL |
|||
|
Stem cell |
40190ES08/10 |
5 mL / 10 mL |
Mainly used for hESC/iPSC stem cell culture |
|
|
Organoid-Specific |
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES08/10 |
5 mL / 10 mL |
Upgraded organoid matrix gel for normal and tumor tissues with improved culture performance. |