Cell migration, also known as cell crawling, cell movement or cell movement, refers to the directional movement of cells after receiving migration signals or sensing the gradient of certain substances. It is a normal functional behavior of cells in the body. Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix. It is often used to evaluate the ability of tumor cells to metastasize in normal tissues, but normal cells, such as macrophages, also have the same ability. Common methods for analyzing cell migration or invasion ability include Transwell migration, Transwell invasion and cell scratching.
I. Cell scratching experiment
1. Experimental principle
Cell scratching is a relatively simple method to measure cell migration movement. It is similar to the body's wound healing model. On a monolayer of adherent cells cultured in a culture dish or plate in vitro, a micropipette tip or other experimental equipment is used to scratch the central area of cell growth, gently remove the cells in the central part, and then continue to culture the cells until the time set in the experiment. Take out the cell culture plate and observe whether the cells around the scratch grow (repair) to the central scratch area to judge the cell growth and migration ability.
2. Experimental steps
2.1 Cell plating
2.1.1 Discard the original culture medium, add trypsin for digestion, centrifuge at 1300 r/min for 3 min;
2.1.2 Discard the supernatant, add 1 mL of fresh culture medium to resuspend, make a single cell suspension, and count with a hemocytometer;
2.1.3 Add culture medium and dilute it to 5×105 cells/mL (the specific number of different cells is different, adjust according to the growth rate);
2.1.4 Add the diluted cell suspension to a 24-well plate, 1 mL per well, mix well by cross-cross method, and culture in a 37 ℃, 5% CO2 incubator.
2.2 Scratching
2.2.1 Observe under a microscope. When the cells cover the entire well plate, use an empty pipette tip to draw a vertical line in the center of the plate;
2.2.2 Discard the culture medium and add 1 mL PBS to wash away the floating cells;
2.2.3 After discarding the culture medium, add 1 mL of serum-free basal culture medium and place in an incubator for culture.
2.3 Photographing
After the cells are cultured to the predetermined time point, perform optical microscopic imaging and save the original picture.
3. Example of experimental results
After the scratch, the cells in the experimental group continued to be cultured for 24 h. The cells migrated to the middle faster than those in the control group, indicating that the cells had a stronger migration ability.
II. Transwell migration or invasion assay
1. Experimental principle
Transwell, as the name implies, is a "perforation assay". Its basic principle is to place a small chamber into a culture plate. The small chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper and lower layers of culture fluid are separated by a polycarbonate membrane. The upper culture fluid is added to the upper chamber, and the lower culture fluid is added to the lower chamber. When cells are planted in the upper chamber, due to the permeability of the membrane, the components in the lower culture fluid can affect the cells in the upper chamber, so that the effects of the components in the lower culture fluid on cell growth, movement, etc. can be studied. The difference between Transwell migration and invasion assays is that the latter requires the laying of matrix gel on the bottom of the upper chamber.
2. Experimental steps
2.1 Transwell migration
2.1.1 Aspirate the original culture medium in the culture dish and wash with 2 mL PBS;
2.1.2 Discard PBS, add 2 mL trypsin for digestion for 3 min, transfer to EP tube by pipetting, centrifuge at 1000 rpm for 5 min;
2.1.3 Discard the supernatant, add 1 mL serum-free culture medium and mix into cell suspension, and count the cells.
2.1.4 Add 200 μL of cell suspension to the chamber and 500 μL of serum-containing culture medium to the 24-well plate;
2.1.5 Culture in the incubator for 24 h;
2.1.6 Take out the chamber and discard the culture medium, wash twice with PBS;
2.1.7 Wipe off the cells in the inner layer of the chamber with a cotton swab and fix with tissue fixative for 10 min;
2.1.8 Air dry and soak with crystal violet dye for 10 min;
2.1.9 Wash off the excess dye with PBS, count and take pictures after air drying (100×).
2.2 Transwell invasion
2.2.1 Melt the matrix gel at 4 ℃ overnight;
2.2.2 Dilute the matrix gel with 4 ℃ pre-cooled serum-free medium to a final concentration of 1 mg/mL (operate on ice);
2.2.3 Add 100 μL of diluted matrix gel vertically to the center of the bottom of the chamber, incubate at 37 ℃ for 4 hours to form a dry gel;
2.2.4 Aspirate the original culture medium in the culture dish and add 2 mL PBS to wash;
2.2.5 Discard PBS, add 2 mL of trypsin for digestion for 3 min, transfer to an EP tube by pipetting, and centrifuge at 1000 rpm for 5 min;
2.2.6 Discard the supernatant, add 1 mL of serum-free medium to mix into a cell suspension, and count the cells;
2.2.7 Add 200 μL of cell suspension to the chamber, about 5×104 cells (diluted with empty culture medium), and add 500 μL of culture medium containing 10% serum to the lower chamber;
2.2.8 Place in a 37 ℃ incubator and culture for 24 hours;
2.2.9 Take the chamber, discard the culture medium, and wash twice with PBS;
2.2.10 Wipe off the cells in the inner layer of the chamber with a cotton swab, and fix with 4% tissue fixative for 10 minutes;
2.2.11 Air dry, soak in 0.1% crystal violet dye for 10 minutes;
2.2.12 Wash off the excess dye with PBS, air dry, take photos and count (100×).
3. Example of results
The cells that pass through the membrane are stained purple by crystal violet, and the greater the number of cells, the stronger the migration/invasion ability of the cells.
References
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[3] Cory G. Scratch-wound assay. Methods Mol Biol. 2011;769:25-30.
[4] Marshall J. Transwell(®) invasion assays. Methods Mol Biol. 2011;769:97-110.
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