Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Dilution and Plating: Thaw Ceturegel™ Matrix on ice (overnight at 4°C). Dilute the matrix with pre-chilled serum-free medium at a 1:5 ratio and mix gently. Immediately pipette an appropriate volume (approx. 60–100 µL, enough to cover the membrane) into the upper chamber of the Transwell insert.
- Gelation: Place the coated inserts in a 37°C incubator for 1–2 hours to allow the matrix to polymerize completely. (Note: 4°C is not recommended for gelation; 37°C incubation is the standard.)
- Hydration: Before use, add 100–200 µL of serum-free medium to each upper chamber and incubate at 37°C for 30 minutes. Carefully aspirate the medium afterward.
II. Cell Preparation
- Cell Suspension: Harvest U87 cells in the logarithmic growth phase using trypsin and stop digestion with serum-containing medium. Centrifuge and discard the supernatant. Wash gently with PBS 1–2 times. Finally, resuspend the cells in serum-free medium and adjust the density to 5.0×10⁵ cells/mL (to achieve a target of 5×10⁴ cells per well).
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 500 µL of complete medium containing 10% FBS to the lower chamber of the 24-well plate as the chemoattractant.
- Seeding and Bubble Removal: Carefully add 100 µL of the cell suspension (containing 5×10⁴ cells) to the upper chamber.
【Note】: Air bubbles between the lower chamber medium and the insert membrane will weaken or eliminate the chemotactic gradient. Be vigilant during pipetting. If bubbles appear, gently lift the insert to release them before reseating it securely in the plate.
- Incubation: Place the assembled plate in a standard 37°C, 5% CO₂ incubator for 24 hours. (Note: Optimize time based on cell invasiveness and drug effects.)
IV. Fixation, Staining, and Analysis
- Fixation: After incubation, remove the insert and discard the medium. Use a moist cotton swab to gently wipe the top surface of the membrane to thoroughly remove non-migrating/non-invading cells. Wash the insert twice with PBS. Place the insert in a container with 4% paraformaldehyde and fix at room temperature for 30 minutes.
- Drying: Discard the fixative and air-dry the insert in a ventilated area at room temperature until the membrane becomes opaque and free of visible water spots (avoid over-drying).
- Staining and Washing: Add 0.1% Crystal Violet staining solution and stain at room temperature for 20 minutes. Discard the stain and wash the insert three times with PBS to remove background color.
- Counting and Analysis: Air-dry the inserts and observe under an optical microscope. Randomly select at least 5 fields of view (avoiding edges) to photograph and count the stained cells that have penetrated the membrane.
- Result Interpretation: As the drug concentration increased, 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. Incubation: Optimize invasion time via pilot experiments (typically 12–48 hours).
5. Wiping: Wipe the upper chamber gently and consistently (once) to avoid damaging the membrane.
6. Counting: Count at least 5 non-overlapping fields per insert (avoiding edges).
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 |