Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Pre-chilling: Place Ceturegel™ Matrix on ice and thaw at 4°C overnight. Simultaneously, pre-chill consumables such as pipettes, tips, and Transwell inserts in a 4°C refrigerator overnight.
- Matrix Dilution and Coating: On ice, dilute the matrix to 200–300 μg/mL using pre-chilled serum-free medium and mix thoroughly with pre-chilled tips. Pipette 200 μL of the dilution along the sidewall of the upper chamber of the Transwell insert. Gently tap the side of the plate to ensure the liquid surface is as flat as possible.
- Gelation: Place the coated inserts in a 37°C incubator and let stand for 0.5–4 hours. After gelation, carefully aspirate any residual matrix from the upper chamber.
II. Cell Preparation
- Serum Starvation: Harvest healthy U251 cells and subject them to serum starvation in serum-free medium for 24 hours.
- Cell Suspension Preparation: Digest the starved cells with trypsin, centrifuge to collect, and wash twice with PBS to thoroughly remove residual FBS. Finally, resuspend the cells in serum-free medium and perform cell counting.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600 μL of complete medium containing 15% FBS and antibiotics (double antibody) to the lower chamber of the 24-well plate.
- Seeding Cells: Place the matrix-coated Transwell inserts into the wells using forceps. Seed 5×10⁴ cells per insert (i.e., 200 μL of cell suspension, serum-free) along the sidewall. Gently tap the side of the plate to flatten the liquid surface.
- 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 48 hours (determined based on preliminary experiments).
IV. Fixation, Staining, and Analysis
- Fixation: After incubation, remove the Transwell insert and discard the medium from the upper chamber. Gently place the insert into a new 24-well plate and wash once with PBS.
- Critical Step: Use a cotton swab or cotton ball moistened with PBS to very gently wipe the residual matrix gel and non-invaded cells from the inside of the insert.
- Transfer the insert to another new 24-well plate using forceps. Add 600 μL of 4% paraformaldehyde and fix at room temperature for 20 minutes.
- Staining and Rinsing: After fixation, discard the fixative and wash with PBS at room temperature for 5 minutes. Add 0.1% Crystal Violet staining solution and stain at room temperature for 30 minutes. After staining, wash the insert by soaking in PBS for 10 seconds.
- Observation and Counting: Air-dry the inserts in a fume hood to remove residual water. Randomly select 4–5 fields of view under a microscope to take photos. Count the number of cells that have penetrated the membrane in each field.
- Result Interpretation: Following drug treatment, the number of migrated cells decreased, indicating that the drug inhibits the invasion of U251 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 |