Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Dilution: Thaw the Ceturegel™ Matrix on ice (overnight at 4°C). Keep all materials on ice. Dilute the matrix with pre-chilled serum-free medium at a 1:8 ratio.
- Coating: Using a pre-chilled pipette tip, carefully pipette approximately 60 μL of the diluted matrix into the upper chamber of the Transwell insert. Gently rock the insert to ensure the membrane is evenly covered. Avoid generating bubbles.
- Gelation: Place the coated inserts in a 37°C incubator for 1–3 hours to allow the matrix to polymerize into a gel.
[Note]: Do not exceed 8 hours to prevent excessive dehydration and cracking of the gel.
II. Cell Preparation and Starvation
- Serum Starvation: 12–24 hours before the experiment, replace the medium of healthy 231 breast cancer cells with serum-free medium to eliminate background stimulation.
- Cell Suspension: Harvest the starved cells using trypsin. Resuspend and centrifuge to collect the cells. Wash gently with PBS 1–2 times to remove residual trypsin and serum. Finally, resuspend the cells in serum-free medium and adjust the density to 1×10⁵ – 5×10⁵ cells/mL (optimize based on cell status).
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.
[Note]: Add liquid carefully to avoid bubbles. Ensure the bottom membrane contacts the liquid to maintain the chemotactic gradient.
- Insert Assembly and Seeding: Place the solidified, matrix-coated inserts into the 24-well plate using forceps. Check for and remove any bubbles between the insert and the well bottom. Carefully add 200 μL of the prepared cell suspension to the upper chamber. Avoid scratching the matrix layer.
IV. Incubation, Fixation, Staining, and Analysis
1. Incubation: Incubate the plate in a standard 37°C, 5% CO₂ incubator for 24–48 hours (optimize time based on invasion rate).
2. Fixation and Staining:
- Removal and Wiping: After incubation, remove the insert and aspirate the medium from the upper chamber. Use a moist cotton swab to gently wipe the inside (top surface) of the insert to remove non-invading cells. Be careful not to disturb the cells on the bottom surface.
- Fixation: Immerse the insert in 4% paraformaldehyde for 15–30 minutes at room temperature.
- Washing: Wash the insert twice with PBS.
- Staining: Stain the invaded cells with 0.1% Crystal Violet for 10–20 minutes.
- Rinsing: Gently rinse with PBS several times until the rinse solution is clear.
3. Counting and Analysis: Air-dry the inserts and observe under a light microscope. Randomly select at least 5 fields of view (avoiding edges) to photograph and count the stained cells.
- Interpretation: A decrease in cell count in the knockdown group vs. control indicates inhibited invasion; an increase in the overexpression group indicates promoted invasion.
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 |