Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing: One day before the experiment, transfer Ceturegel™ Matrix from -20°C to ice and place it in a 4°C refrigerator overnight to thaw completely into a liquid state.
- Coating and Solidification: Perform all steps on ice to prevent premature polymerization. Coat the upper surface of the Transwell insert membrane evenly with an appropriate amount of diluted Ceturegel® Matrix. Place the inserts in a laminar flow hood and air-dry at room temperature for approximately 3 hours to allow the matrix to adhere firmly to the membrane.
【Note】: This "air-drying" method differs from the standard 37°C gelation protocol. Please verify the specific procedure based on the matrix product manual.
II. Cell Preparation
- Digestion and Washing: Harvest HCT116 cells in the logarithmic growth phase via standard trypsin digestion. Centrifuge to collect cells and wash gently with PBS 1–2 times to thoroughly remove residual serum.
- Cell Suspension: Resuspend the cells in serum-free medium and adjust the density precisely to 5×10⁵ cells/mL using a cell counter.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600 µL of complete medium containing 10% FBS to each well of the lower chamber in a 24-well plate to serve as the chemoattractant.
- Seeding: Carefully add 200 µL of the cell suspension (containing 1×10⁵ cells) to the upper chamber of the Transwell insert.
- Avoid Bubbles: Pay special attention during operation to ensure there are no air bubbles between the lower chamber medium and the bottom membrane, as these will severely impair the chemotactic effect.
- Incubation: Place the assembled plate in a standard 37°C, 5% CO₂ incubator and incubate for 12, 24, and 48 hours respectively to obtain invasion data at different time points.
IV. Fixation, Staining, and Analysis
- Fixation: At the predetermined time points, remove the Transwell inserts and discard the culture medium. Rinse the inserts twice with PBS.
- Critical Step: Use a moist cotton swab to carefully wipe the inner surface of the upper membrane to thoroughly remove non-penetrated cells. Place the inserts into a 24-well plate containing 4% paraformaldehyde (or 95% alcohol) and fix at room temperature for 20 minutes.
- Staining: After fixation, discard the fixative and stain directly with 0.1% Crystal Violet solution (or other nuclear stains) at room temperature for 15 minutes.
- Rinsing and Observation: After staining, gently rinse the inserts with running water or PBS to remove excess stain, and air-dry at room temperature.
- Counting and Analysis: Observe the inserts under an inverted microscope. Randomly select 5 fields of view for each sample to take photos. Count the number of cells that have penetrated the membrane in each field and calculate the average value for the sample at that time point.
- Result Interpretation: As the incubation time progressed, the number of migrated cells gradually increased.
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 |