Interventional effect of cyanidin-3-O-glucoside on reproductive toxicity of 1,3-dichloro-2-propanol
A technology of glucoside and cyanidin, applied in the field of medicine, can solve problems such as hazards, life-threatening, and weight loss of organisms, and achieve significant research results
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Embodiment 1
[0038] Example 1 MTT method to screen the concentration of 1,3-DCP on R2C cells and the concentration of C3G intervention
[0039] 1. MTT method to screen the concentration of 1,3-DCP on R2C cells
[0040] (1) Experimental method
[0041] 1) Cells were cultured to the logarithmic phase, digested with trypsin, centrifuged at 1500r / min for 5min, and diluted to 4×10 4 cells / mL, spread on a 96-well plate, and add about 200 μL of suspension to each well;
[0042] 2) After 24 hours, add different concentrations of 1,3-DCP to the corresponding groups, the concentration of each group is 1, 2, 4, 6, 8 mmol / L, 5 replicate wells in each group, and each group is repeated 3 times , and set a cell-free blank group;
[0043] 3) After 24 hours, absorb the liquid, add 10 μL of 5 mg / mL MTT reagent to each well, and place in a 37°C incubator;
[0044] 4) After 4 hours of MTT action, remove the liquid in the well and replace it with 200 μL of DMSO reagent, place the 96-well plate on a shake...
Embodiment 2
[0055] Embodiment 2 MTT method measures the inhibitory effect of C3G on 1,3-DCP in terms of cell activity
[0056] 1. Experimental method
[0057] (1) Cell culture and seeding Cell culture to the logarithmic phase, digest with trypsin, centrifuge at 1500r / min for 5min, and dilute the precipitate to 4×10 4 cells / mL, spread on a 96-well plate, and add about 200 μL of suspension to each well.
[0058] (2) 24 hours after inoculating cells in a 96-well plate, add C3G solution diluted with F12 culture medium (except the control group) to the corresponding groups in one of the 96-well plates, so that the final concentration of C3G in each well of each group is 5, 10, 20, 40 μmol / L. Add C3G solution and 1,3-DCP solution diluted in F12 culture medium to another 96-well plate, except for the control group, make the C3G in other groups be 5, 10, 20, 40 μmol / L respectively, 1,3-DCP in each well of each group The concentration of 3-DCP is 2mmol / L. The settings of the duplicate wells ...
Embodiment 3
[0063] Example 3 Intervention of C3G on morphological changes of R2C cells caused by 1,3-DCP detected by fluorescence microscopy
[0064] 1. Experimental method
[0065] (1) After resuspending the logarithmic phase cells and diluting the cells, inoculate 1 mL (approximately 20,000 cells) in each well of a 6-well plate.
[0066] (2) After 24 hours, add C3G solution and 1,3-DCP solution diluted with F12, except the first group, so that the C3G added in each well of each group is 5, 10, 20, 40 μmol / L, each group The 1,3-DCP concentration of the wells was 2 mmol / L.
[0067] (3) Stimulate each concentration of C3G and 1,3-DCP for 24 hours, check the cell morphology with a fluorescence microscope and take pictures.
[0068] 2. Observation of cell morphology by fluorescence microscope Figure 4 shown. The results showed that, compared with the cells in the control group, under the action of 2 mmol / L 1,3-DCP, the shape of R2C cells was significantly smaller and the number of cel...
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