Application of uabain for enhancing cellular sensitivity of non-small cell lung cancer (NSCLC)
A non-small cell lung cancer, sensitive technology, applied in medical preparations containing active ingredients, organic active ingredients, peptide/protein ingredients, etc., can solve the problem of insufficient tumor cell selectivity, loss of the best time for surgery, normal tissue and cell damage
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Embodiment 1
[0020] Example 1 Annexin V / PI Flow Cytometry Detection of A549 Cell Apoptosis Caused by the Combination of TRAIL and Ouabain Non-small cell lung cancer cells A549 were cultured in DMEM+10% fetal bovine serum culture medium, the culture condition was 37°C, 5%CO 2 moist sterile environment. When the cells grow to 80%-90% confluent, they are washed twice with PBS and digested with 0.25% trypsin. According to the cell volume 6×10 4 Each well was seeded in a 24-well cell culture plate. After the cells adhere to the wall and grow completely, add the drugs respectively TRAIL 10ng / ml, TRAIL 10ng / ml+ouabain 100nM, TRAIL 20ng / ml, TRAIL 20ng / ml+ouabain 100nM, TRAIL 50ng / ml, TRAIL 50ng / ml+ouabain For 100nM, TRAIL 100ng / ml, TRAIL100ng / ml+ouabain 100nM, TRAIL 200ng / ml, TRAIL 200ng / ml+ouabain 100nM, TRAIL400ng / ml, TRAIL 400ng / ml+ouabain 100nM, 3 wells in each group ( figure 1 ), or TRAIL100ng / ml+ouabain 10nM, ouabain 10nM, TRAIL 100ng / ml+ouabain 20nM, ouabain 20nM, TRAIL100ng / ml+ouabain ...
Embodiment 2
[0023] Example 2 Fluorescence Microscopy Detection of A549 Cell Nuclear Fragmentation Caused by the Combination of TRAIL and Ouabain Non-small cell lung cancer cells A549 were cultured in DMEM+10% fetal bovine serum culture medium, and the culture conditions were 37°C, 5% CO 2 moist sterile environment. When the cells grow to 80%-90% confluent, they are washed twice with PBS and digested with 0.25% trypsin. According to the amount of cells 1×10 5 Each well was seeded in a 6-well cell culture plate. Pre-place the sterilized cover glass surface in the culture plate, and when the cells grow to 60% adherent, add drug treatment, respectively control group, TRAIL 100ng / ml, TRAIL 100ng / ml+ouabain 50nM, TRAIL 100ng / ml , TRAIL 100ng / ml+ ouabain 50nM, each group repeated 3 wells ( image 3 ). After 12 hours of treatment, the glass slides were taken out, washed twice with cold PBS, and a few drops of DAPI (1 μg / ml) were added, observed on a fluorescent microscope, and filmed.
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Embodiment 3
[0025] Example 3 Effects of TRAIL and ouabain on the activity of caspase3 hydrolase Non-small cell lung cancer cells A549 were cultured in DMEM+10% fetal bovine serum culture medium, the culture conditions were 37°C, 5% CO 2 moist sterile environment. When the cells grow to 80%-90% confluent, they are washed twice with PBS and digested with 0.25% trypsin. According to the cell volume 6×10 4 Each well was seeded in a 24-well cell culture plate. Add drug treatment, respectively control group, TRAIL100ng / ml, TRAIL 100ng / ml+ouabain 50nM, TRAIL 100ng / ml, TRAIL 100ng / ml+ouabain 50nM, each group repeated 3 wells ( Figure 4 ). After 12 hours of treatment, the cells were digested with trypsin, 1-2 μl of FITC-labeled DEVD.fmk was added, incubated on ice for 30 minutes, washed twice with PBS, and the fluorescence intensity of FITC was analyzed by flow cytometry.
[0026] Results: When the cells are in the state of apoptosis, it will cause the activation of caspase3, so that many pro...
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