Experimental reagent

DMEM medium

Fetal bovine serum

PBS

BD24 cell culture plate

Raininpipettips, 1ml

Glutaraldehyde

Ethanol

purple crystal

Laboratory equipment

Cell culture instrument: 37 ° C and 5% CO 2

Experimental Materials

Person MDA-MB-231cell

Experimental procedure

1. Cells were grown in DMEM medium containing 10% FBS.

2. The cells are seeded at a certain density into a 24-well cell culture plate. After 24 hours of growth, the monolayer cell fusion should reach 70-80%.

3. Do not change the medium. Gently scratch the cells between the single-layer culture cells with a new 1 ml pipette tip. The scratches are traversed through the holes, and the tips are as perpendicular as possible to the bottom of the plate holes. Do not tilt. The gap produced in this way is equal to the outer diameter of the tip of the tip. The distance of the Gap can be adjusted with different types of tips. The scratches are in a straight line in the same direction.

4. Make another scratch in the direction perpendicular to the first scratch, and each hole is scratched into a crisscross pattern.

5. After scratching, gently clean the plate well twice with medium to remove exfoliated cells.

6. Add fresh medium to each well.

7. (The medium contains certain ingredients, such as chemicals that inhibit/promote cell migration and/or proliferation).

8. Cells grow for 48 hours (or as needed).

9. Wash the cells twice in 1 x PBS and fix for 30 minutes using 3.7% paraformaldehyde.

10. 0.1% crystal violet (2% ethanol dissolved) was stained for 30 minutes.

11. Dyed monolayer cells were selected for different fields of view and photographed with a microscope. The distance of the gap can be measured by Photoshop or ImagJ software. In order to reduce the variability of the experimental results, it is recommended to select multiple visual field observations per well, with multiple repetitions per group.

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