Cell invasion

Put the cell chamber (Transwell chamber) into the culture plate, the chamber is called the upper chamber, and the inside of the culture plate is called the lower chamber, the upper chamber contains the upper layer culture solution, the lower chamber contains the lower layer culture solution, and the upper and lower layer culture solution is separated by a polycarbonate membrane. We planted the cells in the upper chamber. Due to the permeability of the polycarbonate membrane, the components in the lower culture medium can affect the cells in the upper chamber, so that we can study the influence of the components in the lower culture medium on cell growth and movement^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Thaw the Matrigel solution overnight at 4℃.

2. Apply 25 µL of Matrigel solution on the lower surface of the Transwell membrane.

3. Incubate Transwell and Matrigel at 37℃ for 30 minutes to form a gel.

4. Add 500 µL of medium (with or without chemoattractant) to each well of a 24-well plate (e.g., for melanoma cell transwell experiments, use 10% fetal calf serum as chemoattractant).

5. Using sterile forceps, transfer the transwell inserts into each well of a 24-well plate already filled with medium (with or without chemoattractant).

6. Add 400 µL of cell suspension (finally confluent 50-60%) to the upper chamber of the transwell.

7. Incubate at 37℃ and 5% CO₂ for 20-24 hours.

8. Add paraformaldehyde (PFA) at a final concentration of 4% in culture medium to both sides of each transwell insert and fix the cells for 15 min at room temperature.

9. Gently aspirate the medium containing 4% PFA.

10. Wash the transwell insert twice with sterile 1X PBS on both sides of the membrane to remove debris, unattached cells, and excess fixative.

11. Stain cells by incubating transwell inserts (with or without Matrigel) with Hoechst for 15 min at RT in the dark (crystal violet or hematoxylin can also be used).

12. Wash the transwell insert twice with sterile 1X PBS to remove excess Hoechst solution.

13. Imaging: Using a fluorescence microscope covering the entire surface, capture some images of the Transwell-inserted membrane.

14. Data analysis: use open source software such as ImageJ to open and analyze the acquired images.

1. Matrigel solidifies easily at room temperature, and the entire glue laying process needs to be performed on ice.

2. The concentration of Matrigel is also one of the factors that affects cell migration and invasion. Therefore, the concentration and dilution ratio of Matrigel need to be adjusted according to the information provided by the supplier and the specific experimental conditions. If necessary, a pre-experiment can be set up to explore the optimal concentration and dilution ratio.

3. When spreading glue, try to add it vertically to the bottom of the chamber and in the middle of the bottom of the chamber to avoid the generation of air bubbles.

4. When sucking out the unbound Matrigel in the chamber, make sure that the tip of the pipette does not scratch the surface of the gel layer.

5. Different cells have different migration and invasion abilities. A series of cell density gradients can be set to explore the appropriate cell seeding density.

6. Bubbles are often generated during the placement of the chamber. Once bubbles are generated, the chemotactic effect of the lower culture medium will be weakened or even disappear, so special attention must be paid. Once bubbles appear, the chamber needs to be lifted up or removed with a pipette tip, and then placed again after removing the bubbles.

7. 1-2 h after seeding the cells, check the culture plate to ensure that no large bubbles have formed. However, sometimes very small air pockets are observed, but they do not affect cell migration and invasion. They can also be removed by gently tapping the well plate.

8. When cleaning the chamber with PBS, avoid physically touching the bottom of the chamber. You can prepare several sterile beakers or petri dishes, pour an appropriate amount of PBS, and rinse them in sequence to improve efficiency.

9. The chamber needs to be properly dried before taking pictures. Moisture will affect the field of view under the microscope.