Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Dilution: Thaw Ceturegel™ Matrix completely at 4°C overnight. Perform all operations on ice. Dilute with pre-chilled serum-free DMEM at a 1:8 ratio and mix gently.
- Coating and Gelation: Immediately use a pre-chilled tip to pipette 50 μL of the diluted matrix onto the polycarbonate membrane of the Transwell insert. Spread evenly and avoid air bubbles. Place the insert in a 37°C, 5% CO₂ incubator for 4 hours to allow the matrix to polymerize completely, forming an artificial basement membrane.
II. Cell Preparation
- Cell Collection: Harvest AN3CA cells and clinical primary EC cells in the logarithmic growth phase.
- Cell Suspension Preparation: Resuspend cells in serum-free medium. Count and adjust the cell concentration to 5×10⁵ cells/mL.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600 μL of corresponding medium containing 10% Fetal Bovine Serum to the lower chamber of the 24-well plate (serving as the chemoattractant).
- Seeding and Drug Treatment: Add 200 μL of the cell suspension (containing 1×10⁵ cells) to the upper chamber of the Transwell insert. Simultaneously, add the corresponding concentration of the drug to the upper chamber according to the experimental grouping.
- 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 block the chemotactic effect.
- Incubation: Place the assembled plate in a 37°C, 5% CO₂ incubator and incubate for 24 hours.
IV. Fixation, Staining, and Analysis
- Fixation: After incubation, remove the Transwell insert and discard the medium from both chambers.
- Critical Step: Use a moist cotton swab to very gently wipe the membrane surface of the upper chamber to thoroughly remove non-invaded cells and residual matrix gel.
- Immerse the insert in 4% paraformaldehyde and fix at room temperature for 30 minutes. After fixation, wash the insert twice with PBS.
- Staining and Rinsing: Immerse the insert in 0.1% Crystal Violet staining solution and stain at room temperature for 20 minutes. After staining, wash the insert 3 times with PBS to remove excess dye.
- Counting and Analysis: Air-dry the insert appropriately and observe under an inverted microscope using a 10× objective. Randomly select 5 fields of view per well to photograph and count the stained cells that have penetrated the membrane.
- Result Interpretation: Following drug treatment, the number of migrated cells decreased, indicating that the drug inhibits the invasion of AN3CA 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 |