Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Pre-chilling: Thaw Ceturegel™ Matrix in a 4°C refrigerator overnight. Perform all experimental steps on ice and use pre-chilled pipette tips and centrifuge tubes to prevent premature gelation.
- Matrix Dilution and Coating: Dilute the matrix with pre-chilled serum-free medium at a 1:8 ratio, keeping it on ice at all times. Add approximately 50 μL of the diluted matrix to the upper chamber of each Transwell insert (8 μm pore polycarbonate membrane). Gently rock the insert to ensure even coverage of the entire membrane surface, taking care to avoid air bubbles.
- Gelation: Place the coated inserts in a 37°C, 5% CO₂ incubator and incubate for 30–60 minutes until the matrix is completely solidified.
II. Cell Preparation
- Drug Treatment: Harvest 4T1 cells in the logarithmic growth phase (70%–80% confluence) and treat with nanomaterials in 6-well plates according to the established grouping scheme for 24 hours.
- Cell Suspension Preparation: After treatment, gently wash the cells once with PBS. Digest with trypsin, collect the suspension, and centrifuge at 1000 rpm for approximately 3 minutes. Discard the supernatant, resuspend in serum-free medium, and pipette gently to ensure a uniform single-cell suspension. Count the cells and adjust the concentration to approximately 2×10⁵ cells/mL, ensuring consistent cell numbers across all groups.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600–800 μL of complete medium containing 10% FBS to the lower chamber of the 24-well plate to serve as the chemoattractant source, establishing a stable gradient between the serum-free upper chamber and the serum-containing lower chamber.
【Critical Note:】 Add liquid slowly along the well wall to avoid vibrating the Transwell or splashing liquid onto the upper membrane, which could destabilize the gradient.
- Seeding Cells: Mix the cell suspensions of each group thoroughly. Add 200 μL (containing approximately 4×10⁴ cells) to each Transwell upper chamber. Place the insert gently to avoid disturbing the matrix layer or creating bubbles, and ensure the liquid surface is evenly covered.
- Incubation: Place the assembled plate in a 37°C, 5% CO₂ incubator for 24 hours. Minimize movement and vibration during incubation.
IV. Fixation, Staining, and Analysis
- Removal of Non-invaded Cells: After incubation, carefully discard the medium from both chambers. Wash gently with PBS 1–2 times. Use a moist cotton swab to gently wipe the upper membrane surface in a single direction to remove non-invaded cells. Ensure consistent pressure to avoid puncturing the membrane or scraping off the matrix.
- Fixation: Add 4% paraformaldehyde to the lower chamber and fix for 15–20 minutes. Discard the fixative and wash twice with DPBS.
- Staining and Rinsing: Add 0.1% Crystal Violet staining solution and stain for 20 minutes. After staining, gently rinse with running water (or soak the insert in a PBS beaker) until the background is clear. Air-dry the Transwell in a ventilated area at room temperature until the membrane is dry.
-
Counting and Analysis:
- Place the bottom surface of the Transwell membrane (where the migrated cells are located) under a microscope.
- Randomly select 5 non-overlapping fields of view per well for photography. Count the number of migrated cells in each field, calculate the average for each well, and then compute the group averages to determine statistical differences.
- Result Interpretation: Following treatment, the number of migrated cells decreased, indicating that the treatment inhibits the invasion of 4T1 cells.
Figure: Cell invasion assay results.
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 |