Experiment Products Used
|
Product Name |
Cat.NO. |
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40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Preparation: Transfer the matrix from -20°C to ice the night before the experiment, then place it in a 4°C refrigerator to thaw overnight. Before the experiment, pre-chill the required pipette tips, EP tubes, etc.
- Coating and Gelation: Perform all steps on ice. Dilute the thawed Ceturegel™ Matrix with pre-chilled serum-free medium at a 1:8 ratio and mix well. Immediately pipette 80 μL of the dilution and spread it evenly on the polycarbonate membrane surface of the Transwell insert. Place the insert in a 37°C incubator and incubate for 2 hours to allow the matrix to completely solidify into a thin film.
- Basement Membrane Hydration: Add 200 μL of serum-free medium to the upper chamber of the insert with solidified matrix, and hydrate in a 37°C incubator for 30 minutes. Carefully aspirate and discard the medium before use.
II. Cell Preparation and Treatment
- Serum Starvation: Harvest CT26 cells in the logarithmic growth phase and subject them to serum starvation in serum-free medium for 12 hours.
- Cell Suspension Preparation: Digest the cells, centrifuge (1000 rpm, 3 minutes) to collect, and wash once with PBS. Resuspend the cells in serum-free medium and adjust the density to 1.5×10⁵ cells/mL.
III. Cell Seeding and Co-culture
- Seeding Cells: Aspirate and discard the excess liquid from the upper chamber of the insert after hydration. Take 400 μL of the cell suspension (containing 6×10⁴ cells) and add it to the upper chamber of the Transwell insert.
- Establish Chemoattractant Gradient and Treatment: In the lower chamber of the 24-well plate:
Control Group: Add 600 μL of complete medium containing 10% FBS.
Drug Group: Add 600 μL of medium containing the corresponding concentration of the drug.
[Note]: Avoid generating air bubbles between the lower chamber and the bottom of the insert during operation.
- Incubation: Place the culture plate in a 37°C, 5% CO₂ incubator and incubate routinely for 24 hours.
IV. Fixation, Staining, and Result Analysis
- Fixation: After culture, remove the insert. Use a moist cotton swab to very gently wipe the upper chamber to thoroughly remove non-invaded cells and the matrix gel. Fix the cells with 4% paraformaldehyde for 20 minutes.
- Staining and Washing: Discard the fixative and stain with 1% Crystal Violet staining solution for 20 minutes. After staining, gently wash the insert several times with ultrapure water to remove excess dye.
- Cell Counting and Data Analysis: After drying the insert, observe it under a microscope. Randomly select 5 fields of view, photograph and count the cells that have penetrated to the underside (bottom surface) of the membrane and adhered, and calculate the average for each group.
- Experimental Results: Compared with the control group, the number of transmembrane cells in the drug treatment group was significantly reduced, indicating that the drug can inhibit the invasion of CT26 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 |