Cell line based on low-dose nano-TiO2 induction, and preparation method and application thereof
A technology of inducing cells and low doses, applied in the field of cell biology, can solve the problems of uncertain correlation, limited value, large investment, etc., and achieve the effect of eliminating species differences, strengthening characterization research, and long time period
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Embodiment 1
[0025] Example 1 Construction of cell lines
[0026] Select normal human bronchial epithelial cells Beas2B as the source cells to induce and screen new cell lines. The specific steps are as follows: take out the Beas2B cells in liquid nitrogen, quickly place them in a water bath at 37°C and shake them continuously until the Beas2B cells are rapidly thawed; Beas2B cells were transferred into a culture dish, and RPMI-1640 medium including 10% fetal bovine serum, 1% penicillin and streptomycin was added, and the volume ratio of air and carbon dioxide was 19:1 (v / v), and the temperature was 37°C. Cultivate overnight under the same conditions, change the medium after the Beas2B cells adhere to the wall, and continue to culture. When the Beas2B cells grow to a density of 90%, they are passaged; then select Beas2B cells that are growing vigorously and in good condition, washed twice with PBS, and digested with trypsin until Beas2B The cells became round, and the reaction was termin...
Embodiment 2
[0027] Example 2 Cell Growth Curve Determination
[0028] The cells in good culture state were digested with trypsin and counted. According to the standard of 4000 cells in 200 μL cell suspension per well, the cells were evenly seeded in a 96-well plate, and no cells were planted in the microwells on the four sides of the 96-well plate to culture Liquid seal. Place in an incubator at 37°C, 5% CO 2 Cultivate, add 10uLCCK-8 reagent after 6h, incubate under cell culture conditions for 3-4h, measure the absorbance value at 450nm, and add CCK-8 reagent at the same time for 4 consecutive days, measure the absorbance value at 450nm at the same time, and The absorbance value on the first day was normalized as 100%, and the following days were normalized according to the absorbance value of different cells on the first day, and the growth curve was drawn, see appendix figure 1 , for different low-dose nano-TiO 2 The Beas2B cell proliferation changes after long-term treatment of Be...
Embodiment 3
[0031] Example 3 Cell Migration Test
[0032]Add 500uL of medium to the 24-well plate, put the transwell chamber into it, and incubate in a 37°C incubator for 30min, trypsinize and count the cells in good culture state, and use serum-free medium at a rate of 70,000 per 200uL suspension. A standard of 1 cell was added to the transwell chamber, and placed in an incubator at 37°C, 5% CO 2 Cultured, stained with eosin or 0.5% crystal violet for 10 min after 18 h, and stained with ddH 2 O Rinse, wipe off the upper layer of cells with a cotton swab, and take pictures after drying, as attached Figure 4 As shown in A, nano-TiO 2 The longitudinal migration ability of the cells treated for a long time is reduced; through the scratch test, the cell monolayer cultured in the 6-well plate is scratched with a line through the sterile wall, and the floating cells are washed away, and observed under the microscope at 24 hours and 48 hours , the result is attached Figure 4 As shown in ...
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