Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Reagent Pre-chilling: Before the experiment, place Ceturegel™ Matrix on ice to liquefy at 4°C. Pre-chill the required pipette tips in a 4°C refrigerator.
- Coating and Gelation: Perform all steps on ice. Dilute the matrix with pre-chilled serum-free medium at a 1:8 ratio and mix gently using pre-chilled tips. Immediately pipette 80 μL of the dilution vertically into the upper chamber of the Transwell insert. Gently rock the insert to ensure the matrix covers the membrane surface evenly. Place the insert in a 37°C incubator and incubate for 3 hours to allow the matrix to polymerize completely.
II. Cell Preparation
- Serum Starvation: Select healthy PC-9 cells and subject them to serum starvation in serum-free medium for 24 hours prior to the experiment.
- Cell Suspension Preparation: Digest the cells, wash with PBS, and resuspend in serum-free medium. Adjust the cell density to a range of 1×10⁵ – 5×10⁵ cells/mL via cell counting. (Note: The optimal density should be optimized via pilot experiments based on the specific invasiveness of the cells.)
III. Cell Seeding and Co-culture
- Seeding Cells: Aspirate any unbound liquid from the upper chamber of the Transwell insert. Add 500 μL of the prepared cell suspension to the upper chamber, ensuring even distribution.
- Establish Chemoattractant Gradient: Add 1.5 mL of complete medium containing 10% FBS (or other chemoattractants) to the lower chamber of the culture plate (typically a 6-well plate).
【Critical Note】: Ensure complete contact between the bottom membrane of the insert and the liquid in the lower chamber. Avoid air bubbles, as they will weaken the chemotactic effect.
- Incubation: Place the assembled 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.
- Critical Step: Use a moist cotton swab to gently wipe the membrane surface of the upper chamber to thoroughly remove non-invaded cells and residual matrix gel, retaining only the cells that have penetrated to the bottom surface. Immerse the insert in a container with 4% paraformaldehyde and fix at room temperature for 30 minutes.
- Washing and Staining: Discard the fixative and wash the insert three times with PBS. Add 0.1% Crystal Violet staining solution and stain at room temperature for 10 minutes.
- Rinsing and Observation: Discard the staining solution and wash the insert thoroughly with PBS to remove excess dye.
- Counting and Analysis: Air-dry the insert and observe under an optical microscope. Randomly select at least 5 different fields of view to photograph and count the cells that have penetrated the membrane and adhered to the bottom surface. Calculate the average number of migrated cells for each group (e.g., control vs. experimental).
- Result Interpretation: Compared to the control group, the number of migrated cells in the drug-treated group decreased significantly, indicating that the drug inhibits the invasion of PC-9 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 |