Application of costunolide and/or dehydrocostus lactone in preparation of medicine for treating melanoma
A technology for dehydroxylactone and xylolide, which is applied in the field of biomedicine and can solve the problems of obvious adverse reactions, large trauma and the like
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Embodiment 1
[0049] Example 1 MTT method is used to determine the effect on the proliferation of melanoma cell B16 cells
[0050] Take mouse B16 melanoma cells in the logarithmic growth phase, count the cells under a microscope, and adjust the cell concentration to 1×10 5 cells / mL, inoculated into 96-well plate, 100 μL cell liquid per well, that is, 1×10 4 Each well, after the cells grow on the wall overnight, add 100 μL of fresh medium containing arylactone or dehydrocresinolide, and set the drug concentration to 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and set There is a blank control group without drug addition, and 6 replicate wells are set up in each group, and cultured for 24, 48 and 72 hours respectively, and 20 μL of MTT solution is added to each well, and the cell incubator is further cultivated for 4 hours, the old medium is discarded, and 200 μL DMSO is added to each well, The absorbance value was measured with a microplate reader A570nm.
[0051] Cell proliferation inhibitio...
Embodiment 2D
[0056] Example 2 Observation of Apoptosis by Fluorescence Microscopy after DAPI Staining Cell Nuclei
[0057] Take 3mL1×10 5 cells / mL of B16 cells, inoculated into a 60mm petri dish with glass slides, after growing on the wall overnight, discarded the old medium, and added 3 mL of coycinolide or dehydrocoynelide at a concentration of 25 μM respectively. Fresh culture medium, set blank control group, place in cell culture incubator for 48 hours, wash twice with PBS, add pre-cooled 75% ethanol, fix overnight in 4°C refrigerator, discard fixative, add PBS to wash Three times, add DAPI staining solution, protect from light at 37°C for 30 minutes, wash with PBS, observe cell morphology under a fluorescent inverted microscope, and take pictures for analysis.
[0058] The result is as follows:
[0059] After B16 cells were treated with xylolide, the number of cells observed under the microscope was significantly reduced, and there were shrinkage and digestion phenomena. After DAPI...
Embodiment 3
[0062] Example 3 FCM detection and analysis of cell apoptosis by flow cytometry
[0063] Take 3mL1×10 5 cells / mL of B16 cells were inoculated into a 60mm culture dish, and after growing overnight, the old medium was discarded, and fresh medium containing different concentrations of drugs was added, and the group without adding drugs was set as the control group. Place in the cell incubator and continue to culture for 48 hours, digest the cells with EDTA-free trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash twice with PBS, resuspend the cells in PBS, and take 1×104 resuspended cells, After centrifugation, add 195 μL AnnexinV-FITC conjugate solution and gently resuspend the cells. Add 5 μL AnnexinV-FITC staining solution, mix gently, add 10 μL PI, mix gently. Incubate at room temperature in the dark for 20 min, then place on ice, detect by flow cytometry, and analyze the apoptosis rate.
[0064] The result is as follows:
[0065] Flow cytometry de...
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