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Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Dilution: Thaw Ceturegel™ Matrix on ice (overnight at 4°C). Keep all materials on ice. Dilute the matrix with pre-chilled serum-free medium at a 1:1 ratio. (Note: This ratio yields a high final concentration of approx. 4.5–5 mg/mL, suitable for highly invasive cells or models requiring a thicker barrier.)
- Coating: Use Transwell inserts with an 8 µm pore size. Using a pre-chilled pipette tip, carefully pipette 50 µL of the diluted matrix into the center of the polycarbonate membrane. Gently rock the insert horizontally to ensure the membrane is evenly covered. Avoid generating bubbles.
- Gelation: Place the coated inserts into a 24-well plate rack and incubate at 37°C for 1–2 hours to allow the matrix to polymerize into a gel, forming a simulated ECM layer.
II. Cell Preparation
1. Digestion and Collection: Aspirate the old medium from UMR-106 cells and wash gently with sterile PBS. Digest cells with trypsin. Once cells round up and detach, add complete medium containing serum (or an equal volume of serum-free medium) to stop digestion.
2. Cell Suspension: Transfer the suspension to a centrifuge tube and centrifuge to pellet the cells. Resuspend and wash gently with PBS once to remove residual trypsin and serum. Finally, resuspend the cells in serum-free medium and adjust the density to 1×10⁵ cells/mL.
III. Cell Seeding and Co-culture
1. Establish Chemoattractant Gradient: Add 600 µL of complete medium containing 10% FBS (or other specific chemoattractants) to the lower chamber of the 24-well plate to serve as the chemoattractant source.
2. Seeding: Carefully add 200 µL of the prepared cell suspension (approx. 2×10⁴ cells) to the upper chamber of the matrix-coated Transwell insert. Avoid touching the solidified gel layer or creating bubbles.
3. Incubation: Carefully transfer the assembled plate to a 37°C, 5% CO₂ incubator and incubate for 24 hours.
IV. Incubation, Fixation, Staining, and Analysis
- Fixation: After incubation, remove the insert. Gently wash the inside of the upper chamber once with sterile PBS to remove unattached cells. Place the insert in a container with 4% paraformaldehyde and fix at room temperature for 10–15 minutes.
- Washing and Staining: Wash the insert twice with PBS. Stain the cells with 0.1% Crystal Violet (or other nuclear stain) for 15–20 minutes at room temperature.
- Rinsing and Wiping: Rinse the insert gently with PBS several times until the rinse solution is clear. Use a moist cotton swab to gently wipe the top surface (matrix side) of the membrane to thoroughly remove non-invading cells. Be careful not to disturb the cells on the bottom surface.
- Counting and Analysis: Air-dry the inserts or observe directly under a light microscope. Randomly select at least 5 fields of view to photograph and count the invaded cells on the bottom membrane. Calculate the average number of cells for each group (e.g., treated vs. control).
- Result Interpretation: An increase in the number of migrated cells in the treated group compared to the control indicates that the treatment promotes the invasion of UMR-106 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 |