Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Dilution: Place Ceturegel™ Matrix in a 4°C refrigerator and thaw overnight. Dilute with pre-chilled DMEM to a concentration of 2.5%. Perform all steps on ice or under low-temperature conditions.
- Coating and Gelation: Pipette 50 μL of the diluted matrix and gently add it to the center of the upper chamber of the Transwell insert. Place the insert in a laminar flow hood and let stand at room temperature for 1 hour to allow the ECM gel to solidify completely.
II. Cell Preparation
Cell Suspension Preparation: Harvest Huh7 cells in the logarithmic growth phase via standard trypsin digestion and centrifuge to collect. Resuspend the cells in DMEM containing 10% FBS, count, and adjust the cell density to 5×10⁴ cells/mL.
III. Cell Seeding and Co-culture
- Seeding Cells: Add 200 μL of the cell suspension (containing 1×10⁴ cells) to the upper chamber of the Transwell insert.
- Establish Chemoattractant Gradient: Add 600 μL of DMEM complete medium containing 15% FBS to the lower chamber of the 24-well plate.
【Critical Note:】 Ensure there are no air bubbles between the bottom membrane of the insert and the liquid in the lower chamber, as bubbles will weaken the chemotactic effect.
- Incubation: Place the plate in a 37°C, 5% CO₂ incubator and incubate for 24 hours. Set up 3 replicate wells for each group.
IV. Fixation, Staining, and Analysis
1. Fixation:
After incubation, carefully aspirate the medium from both the upper and lower chambers.
Add 200 μL of 4% paraformaldehyde to the upper chamber and 600 μL to the lower chamber, respectively. Fix the cells at room temperature for 30 minutes.
2. Drying: Discard the paraformaldehyde and air-dry the insert at room temperature for approximately 5 minutes.
3. Staining: Add 200 μL of 0.1% Crystal Violet staining solution to the upper chamber and 600 μL to the lower chamber, respectively. Stain at room temperature for 40 minutes.
4. Rinsing and Removal:
Remove the Crystal Violet solution and wash the insert with PBS for 5 minutes.
Aspirate the PBS and use a moist cotton swab to very gently wipe the membrane surface of the upper chamber to remove non-invaded cells.
5. Observation and Counting:
- Place the insert under an upright microscope to observe the migrated/invaded cells on the surface of the lower membrane, recording cell size and number.
- Randomly select at least 5 fields of view for photography and counting. Calculate the average value for statistical analysis to quantify the invasion ability of Huh7 cells.
Tips
1. Coating: Keep matrix and tips cold to prevent premature polymerization.
2. Cells: Ensure complete serum starvation to reduce background noise.
3. Bubbles: Verify there are no bubbles between the insert and the lower chamber medium.
4. Wiping: Wipe the upper chamber gently and consistently (once) to avoid damaging the membrane.
5. Counting: Count at least 5 non-overlapping fields per insert.
Related Products
|
Product Type |
Product Name |
Cat. No. |
Specification |
|
Standard Concentration |
40183ES08/10 |
5 / 10 mL |
|
|
40184ES08/10 |
5 / 10 mL |
||
|
Low Growth Factor |
40185ES08/10 |
5 / 10 mL |
|
|
40186ES08/10 |
5 / 10 mL |
||
|
High Concentration |
40187ES08/10 |
5 / 10 mL |
|
|
Ceturegel™ Matrix High Concentration, Phenol Red-Free, LDEV-Free |
40188ES08/10 |
5 / 10 mL |
|
|
Ceturegel™ Matrix High Concentration, GFR, LDEV-Free |
40189ES08/10 |
5 / 10 mL |
|
|
For Stem Cell Applications |
40190ES08/10 |
5 / 10 mL |