Cell scratch (repair) method is a simple and convenient method to measure the migration and repair ability of cells.
On the monolayer adherent cells cultured in vitro or plate, the central region of cell growth was marked with a trace of gun head or other hard objects.Remove the central part of the cells, and then continue to culture cells to the time set by the experiment, and then take out the cell culture plate to observe whether the peripheral cells grow (repair) to the central scratch area,
so as to judge the growth and migration ability of cells.
The experiment usually needs to set up the normal control group and the experimental group. The experimental group is a group with some treatment factors or drugs, exogenous genes, etc. the migration and repair ability of each group of cells can be judged by the repair ability of cells in different groups for the scratch area.
1.Culture plate marking
First, use marker pen on the back of the 6-hole plate, compare with a ruler, and draw horizontal lines evenly, about every 0.5~1cm, across the hole. At least 5 lines per hole. Pay attention not to make the line too thick.
2.Spread cell
About 5×105 cells were added into the pore(The number of different cells varies according to the growth rate of cells).The principle of vaccination was that the fusion rate reached 100% after overnight.
3.Cell lineation
The next day, scratch the cell layer with the gun head or sterile toothpick, perpendicular to the cell plane, along the line drawn on the back of the plate(It is better to use the same gun head or toothpick between different holes).
4.Wash cell
After the scratch, the cells were washed with sterile PBS for 3 times.Wash away the non adherent cells,then the fresh serum-free medium was replaced.
5.Cell culture and observation
The cells were cultured in 5% CO2 incubator at 37 ℃.Then the cells were taken out at appropriate time points, such as 0,6,12,24 h. The width of the scratch was observed and measured by microscope line, and take photos(The specific time depends on the needs of the experiment).
6.Result analysis
After the image J software was used to open the picture, 6 to 8 horizontal lines were randomly drawn to calculate the mean distance between cells.
It can only be applied to epithelial cells and fibroid cells.
Because:(1)These cells have the ability of migration and stronger;(2)Cell polarity, convenient measurement, observation;(3)Cells have strong tolerance to serum-free(At least 24 hours),Therefore, many tumor cell lines are not suitable for scratch.
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