Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Dilution: Thaw Ceturegel™ Matrix on ice (overnight at 4°C). Keep all materials on ice. Dilute the matrix with pre-chilled serum-free MEM medium at a 1:8 ratio (e.g., 100 μL matrix + 800 μL medium) to achieve a final concentration of approximately 1 mg/mL (ensure it does not exceed 3 mg/mL). Mix gently by pipetting immediately after dilution.
- Coating: Immediately use a pre-chilled pipette tip to carefully add 50 μL of the diluted matrix dropwise into the upper chamber of the Transwell insert. Gently rock the insert to ensure the membrane is evenly covered (liquid layer thickness approx. 3 mm). Avoid generating bubbles.
- Gelation: Place the coated inserts in a 37°C incubator for 2–4 hours to allow the matrix to polymerize completely.
- Hydration: Before use, add 200 μL of serum-free MEM medium to each upper chamber and incubate at 37°C for 30 minutes. Afterward, carefully aspirate the medium.
II. Cell Preparation and Starvation
1. Serum Starvation: 12–24 hours before the experiment, replace the medium of healthy SiHa cells (including empty vector controls and overexpression groups) with serum-free MEM medium to eliminate background interference.
2. Cell Suspension: Harvest the starved cells using trypsin and stop digestion with serum-containing medium. Centrifuge and discard the supernatant. Wash the cells gently with PBS buffer 1–2 times. Finally, resuspend the cells in serum-free MEM medium and adjust the density to 2.5×10⁵ cells/mL.
III. Cell Seeding and Co-culture
1. Establish Chemoattractant Gradient: Add 600 μL of MEM complete medium containing 20% FBS to the lower chamber of the 24-well plate as the chemoattractant.
2. Seeding: Carefully add 200 μL of the prepared cell suspension (approx. 5×10⁴ cells) to the upper chamber of the Transwell insert. Avoid creating bubbles.
3. Incubation: Place the assembled plate in a standard 37°C, 5% CO₂ incubator for 48 hours.
IV. Fixation, Staining, and Analysis
- Fixation: After incubation, remove the insert and discard the medium from both chambers. Use a moist cotton swab to gently wipe the bottom surface (matrix side) of the upper chamber to thoroughly remove non-invading cells. Wash the insert twice with Ca²⁺/Mg²⁺-free PBS buffer. Place 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 an appropriate amount of 0.1% Crystal Violet staining solution and stain at room temperature for 30 minutes.
- Rinsing and Drying: Gently rinse the insert with deionized water several times until the wash solution is colorless and free of purple residue. Air-dry the insert at room temperature.
- Counting and Analysis: Observe and photograph the dried inserts under an optical microscope using 10× and 20× objectives. Randomly select at least 5 different fields of view to count the stained cells that have penetrated the membrane.
- Result Interpretation: Compared to the control group, the number of migrated cells in the overexpression group increased significantly, indicating that the gene affects the invasion ability of SiHa 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 |