principle:
Cell wound healing experiments have been widely used and improved by researchers to study the role of various experimental conditions such as gene knockout and chemotherapy in cell migration and proliferation. The experiment is simple in operation, inexpensive, and the experimental conditions are easily improved for different purposes. The basic principle of the method is to make scratches between the monolayer cultured cells to produce a callus area, and then monitor the phenomenon of scratch migration to the cells around the wound, that is, wound healing. Factors that alter cell migration and/or growth can increase or decrease the rate of wound healing. This method can be quantified and is suitable for high throughput screening of automated systems.
Materials and instruments:
1. Person MDA-MB-231cell (ATCC#HTB-26)
2. DMEM medium (Invitrogen #10313-021)
3. Fetal bovine serum (ATCC#30-2020)
4.PBS (Invitrogen #14190-144)
5. BD24 cell culture plate (Fisher#08-772-1H; BD#353226)
6.Raininpipettips, 1ml (Rainin#GPS-L1000)
7. Glutaraldehyde (Sigma-Aldrich #G6257)
8. Ethanol (Sigma-Aldrich #459836)
9. Crystal Violet (Sigma-Aldrich C3886)
10. Cell culture instrument: 37 ° C and 5% CO 2
step:
1. Cells were grown in DMEM medium containing 10% FBS.
2. The cells were inoculated into a 24-well cell culture plate at a certain density. After 24 hours of growth, the degree of 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 1ml tip, the scratches traversing the holes, and the tips of the tips are perpendicular 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. Cell growth for 48 hours (or as needed).
The cells were washed twice with 9.1 x PBS and then fixed with 3.7% paraformaldehyde for 30 minutes.
10.10.1% crystal violet (2% ethanol dissolved) was stained for 30 minutes.
11. Stained monolayer cells were selected for different fields of view and photographed by microscopy. 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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