Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Dilution: Thaw Ceturegel™ Matrix overnight at 4°C. Dilute with pre-chilled serum-free medium at a 1:5 ratio and keep on ice.
- Coating and Gelation: Pipette 100 μL of the diluted matrix onto the polycarbonate membrane of the Transwell insert (8 μm pore size). Ensure even coverage and avoid air bubbles. Place the insert in a 37°C incubator for 1–3 hours to allow the matrix to polymerize completely.
- Hydration: Before use, add 200 μL of serum-free medium to the upper chamber and incubate at 37°C for 30 minutes. Carefully aspirate the medium after hydration.
II. Cell Preparation
- Serum Starvation: Harvest U87 cells in the logarithmic growth phase and subject them to serum starvation in serum-free medium for 24 hours.
- Cell Suspension Preparation: Digest the starved cells and wash twice with PBS to thoroughly remove residual serum. Resuspend the cells in serum-free medium and adjust the density to 1×10⁵ cells/mL.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600 μL of complete medium containing 10% FBS to the lower chamber of the 24-well plate.
- Seeding Cells: Add 200 μL of the cell suspension (containing 2×10⁴ cells) to the upper chamber of the Transwell insert.
【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 plate in a standard 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 culture medium.
- Critical Step: Use a moist cotton swab to gently wipe the membrane surface of the upper chamber to thoroughly remove non-invaded cells. Wash the insert twice with PBS. Immerse the insert in 4% paraformaldehyde and fix at room temperature for 30 minutes.
- Staining and Rinsing: Discard the fixative and immerse the insert in 0.1% Crystal Violet staining solution. Stain at room temperature for 20–30 minutes. After staining, wash thoroughly with PBS to remove excess dye.
- Counting and Analysis: Randomly select at least 5 different fields of view (avoiding the membrane edges), 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 U87 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 |