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). Mix thoroughly using pre-chilled pipette tips. On ice, dilute the matrix with pre-chilled serum-free 1640 medium at a 1:8 ratio and mix immediately.
- Coating and Gelation: Using a pre-chilled tip, pipette 80 μL of the diluted matrix vertically into the upper chamber of the Transwell insert. Gently rock the insert to ensure even distribution. Place the inserts in a 37°C incubator for 3 hours to allow the matrix to polymerize completely.
- Hydration and Check: Aspirate any unbound liquid from the insert. Add 100 μL of serum-free 1640 medium and incubate at 37°C for 30 minutes to hydrate the gel. After hydration, aspirate the medium and check for leakage into the lower chamber. If no leakage occurs, the coating is successful and ready for use.
II. Cell Preparation and Treatment
- Serum Starvation: 12–24 hours before the experiment, replace the medium of healthy B16F10 cells with serum-free medium for starvation.
- Cell Suspension: Harvest the starved cells using trypsin. Stop digestion with 1640 medium containing 1% FBS to create a single-cell suspension. Centrifuge (1000 rpm, 3 min) and discard the supernatant.
- Control Group: Resuspend cells in 1640 medium containing 1% FBS and adjust the density precisely to 2.5×10⁵ cells/mL.
- Experimental Group: Resuspend cells in 1640 medium containing 1% FBS and the specific drug and adjust to the same density.
III. Cell Seeding and Co-culture
- Establish Chemoattractant Gradient: Add 600 μL of 1640 complete medium containing 20% FBS to the lower chamber of the 24-well plate as the chemoattractant.
- Seeding: Place the coated Transwell inserts into the 24-well plate using forceps. Carefully add 200 μL of the cell suspension (containing 5×10⁴ cells) from either the control or experimental group into the corresponding upper chamber.
- Incubation: Place the plate in a standard 37°C, 5% CO₂ incubator and incubate for 48 hours.
IV. Fixation, Staining, and Analysis
- Fixation: After incubation, remove the insert and discard the medium. Use a moist cotton swab to gently wipe the matrix and non-invading cells from the upper chamber. Add 600 μL of 4% paraformaldehyde to a clean well in a 24-well plate, place the insert into it, and fix at room temperature for 20 minutes.
- Washing: Discard the fixative and wash the insert once with PBS.
- Staining: Add an appropriate amount of 0.1% Crystal Violet staining solution to a clean well in a 24-well plate. Place the insert into the solution and stain at room temperature for 20 minutes. Afterward, remove the insert and wash three times with PBS.
- Observation and Counting: Air-dry the insert appropriately and observe under an optical microscope. Randomly select at least 5 fields of view to take photos. Count the number of migrated cells for the control and experimental groups and calculate the average.
- Result Interpretation: Compared to the control group, the number of migrated cells in the drug-treated experimental group decreased significantly, indicating that the drug inhibits the invasion of B16F10 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 |