Experiment Products Used
|
Product Name |
Cat.NO. |
|
40183ES |
I. Preparation of Transwell Basement Membrane
- Matrix Thawing and Dilution: Remove Ceturegel™ Matrix from -20°C, place on an ice box, and dissolve in a 4°C refrigerator overnight. After dissolution, dilute with pre-chilled basal medium at a 1:5 ratio (final concentration not exceeding 3 mg/mL). Perform all steps on ice.
- Coating and Gelation: Pipette the diluted matrix and spread evenly on the upper surface of the Transwell insert, avoiding air bubbles. Place the insert in a 37°C incubator and let stand for 30 minutes to 8 hours (typically 30 minutes to 1 hour is sufficient) to allow the matrix to solidify completely.
- Hydration: Before use, add serum-free medium to the upper chamber and hydrate in a 37°C incubator for 30 minutes. Carefully aspirate the medium after hydration.
【Note】: If performing a migration assay, coating with matrix is not required. However, the inserts must be removed from 75% ethanol in advance and dried upside down in a laminar flow hood.
II. Cell Preparation and Pre-treatment
- Serum Starvation: 12–24 hours before the experiment, replace the medium of healthy cells with serum-free medium to eliminate serum effects.
- Drug Pre-treatment: Treat the cells with the corresponding drug in 6-well plates and incubate in a culture incubator for 48 hours.
III. Cell Seeding and Co-culture
- Cell Suspension Preparation: Aspirate the complete medium from the 6-well plate and wash 1–2 times with PBS. Add trypsin to digest for approximately 1 minute, terminate digestion with serum-containing medium, and pipette to collect cells. Centrifuge at 800 rpm for 3–5 minutes. Discard the supernatant, resuspend in basal medium, count using a hemocytometer, and adjust the cell density to 1×10⁵ cells/mL.
- Counting Formula: Cells/mL = (Cell count in 80 squares / 80) × 400 × 10,000.
- Seeding Cells: Use forceps to place the coated Transwell insert into a 24-well plate. Take a cell suspension containing 1×10⁵ cells and adjust the volume to 200 μL with basal medium, then gently add it to the upper chamber.
- Establish Chemoattractant Gradient: Add 600 μL of complete medium containing serum (as a chemoattractant) to the lower chamber of the 24-well plate.
【Critical Note:】 Ensure there are no air bubbles between the lower chamber medium and the bottom of the insert, as this will block the chemotactic effect.
- Incubation: Place the plate in a 37°C, 5% CO₂ incubator and incubate for approximately 16 hours (specific time should be determined by pilot experiments based on cell migration/invasion ability).
IV. Fixation, Staining, and Analysis
1. Washing and Removal of Non-invaded Cells:
- After incubation, remove the Transwell insert. Add 600 μL of PBS to the 24-well plate, place the insert inside, and wash twice, shaking for 5 minutes each time.
- After the second wash, use a cotton swab dipped in 75% alcohol to very gently wipe the upper layer of the insert to thoroughly remove non-invaded cells.
2. Fixation and Permeabilization:
- Fix the cells on the lower layer of the insert using 4% paraformaldehyde (600 μL/well) by soaking at room temperature for 20 minutes. Wash twice with PBS, 5 minutes each time.
- Add 100% methanol (600 μL/well) to the 24-well plate, immerse the insert for 20 minutes to permeabilize the cell membrane, then wash twice with PBS.
3. DAPI Staining:
- Add 600 μL of DAPI staining solution (diluted 1:100 with PBS) to each well and stain in the dark for 15 minutes. Wash twice with PBS, 5 minutes each time.
- Observation and Photography: Invert the Transwell insert onto a coverslip and observe using an upright fluorescence microscope at 10× or 20× magnification.
4. Data Statistics:
- Count the number of migrated cells in each field of view and calculate the average.
- The invasion inhibition rate is calculated as: Invasion Inhibition Rate = (Control Group Count - Experimental Group Count) / Control Group Count × 100%. This quantifies the effect of drug treatment on cell invasion ability.
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 |