cell invasion assay

Summary

Invasion of a malignant tumor into adjacent tissues is called invasion. Detecting the ability of a tumor to invade is a way to determine the ability of a tumor to metastasize.

Principle

Transwells are placed in 24-well culture plates and are divided into upper and lower chambers. At the bottom of the upper chamber is a polycarbonate membrane, which is permeable and separates the culture fluid in the upper chamber from the culture fluid containing the lower chamber, but the tumor cells can pass through, and the culture fluid in the lower chamber (e.g., chemokines in the serum) attracts the cells in the upper chamber.

Operation method

Cell invasion assay

Principle

Transwells are placed in 24-well culture plates and are divided into upper and lower chambers. At the bottom of the upper chamber is a polycarbonate membrane, which is permeable and separates the culture fluid in the upper chamber from the culture fluid containing the lower chamber, but the tumor cells can pass through, and the culture fluid in the lower chamber (e.g., chemokines in the serum) attracts the cells in the upper chamber.

Materials and Instruments

① Cell samples (cells in good condition)
② Consumables: EP tubes, 24-well plates, 8 μm pore size Transwell chambers, and ice boxes.
③ Reagents: serum-free medium, complete medium with 10%~20% FBS, Matrigen gel, 4% paraformaldehyde, crystal violet stain.

Move

1. Preparation before experiment:

(1) Cells should preferably be starved the day before (i.e., incubate in serum-free medium for 1 night to enhance cell migration).

(2) Melt Matrigen gel overnight at 4 ℃ and transfer it to an ice box before the experiment.

(3) Pre-cool the tip head, EP tube, 24-well plate with Transwell and serum-free medium at 4 ℃, and transfer to the ice box before the experiment.

2. Matrix Gel Spreading: (1) Dilute Matrigen

(1) Dilute Matrigen Gum: Add 8 µL of Matrigen Gum to 64 µL of pre-cooled serum-free medium (1:8 dilution is recommended), and then blow with a tip to mix well.

(2) Spreading: Pipette 60 µL of diluted Matrigen gel into the upper chamber of Transwewll vertically and spread it evenly on the bottom. Do not create air bubbles during the spreading process.

(3) Gelation: Incubate Matrigen gel in an incubator (37 ℃, 5% _CO2 ) for 1~3 h to polymerize the film.

(4) Hydrate the basement membrane, aspirate off the excess liquid in the upper chamber, add 100 µL of serum-free medium into each well, and leave it in the incubator for 30 min.

3. Collect the cells, centrifuge to remove the supernatant, resuspend the cells with serum-free medium and count them. Different cells have different sizes and invasive ability, a concentration gradient can be set to achieve the best results. Preparation of cells in each group: 3 replicate wells for each group. For example, the amount of ¹⁰⁵ cells/200 μl per well is used to prepare 4 duplicate wells (1 additional well), i.e. ^4×105 cells/800 μl.

4. Add 700~800 μl of complete medium containing 10%~20% fetal bovine serum into the lower chamber of the transwell, and add 200 μl of cells prepared in (3) into the upper chamber.

5. Cultivate the cells in the cell culture incubator for 24~48 hours, the details need to be explored in the pre-test, as the invasive ability of each type of cell is not consistent. 6.

6, fixation: 4% paraformaldehyde at room temperature for 30 minutes, crystal violet staining solution at room temperature for 30 minutes, appropriate air-drying, photography, counting, and statistical analysis.

Caveat

1. Adequate preparations must be made prior to the experiment, such as melting Matrigen gel overnight at 4 °C, pre-cooling the tip, EP tube, 24-well plate with transwell chambers, and serum-free medium at 4 °C overnight.

2. Handle the transwell chambers gently and do not blow them violently.Avoid air bubbles.

4. Spread the glue evenly.

5， with or without serum medium labeled clearly, do not use the opposite.

Common Problems

1、When rubbing the cells in the upper chamber with a cotton swab, it is easy to rub the cells that have already passed through, so be careful with the operation.

2、Put the transwell chamber into the 24-well plate, the contact surface appears bubbles.

3、Cells should be in good condition, otherwise migration may not happen.

4, the cell inoculation amount of the problem, too much or too little will affect the results of the experiment, it is recommended to do a concentration gradient of the pre-test.

5, the choice of detection time point, also need to carry out pre-test.

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