Method for establishing hypoxia injury cell model
A technology of cell model and hypoxic injury, which is applied in the field of biomedicine to achieve the effect of low modeling cost and sensitive response to hypoxic stress
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Embodiment 1
[0037] Embodiment one, refer to the attached Figure 2-3 .
[0038] Establishment of hypoxic injury model of rat liver cells (BRL)
[0039] 1. BRL cell culture conditions:
[0040] BRL cells were cultured in DMEM medium containing 10% fetal bovine serum (containing 1×105 IU / L penicillin and 1×105 IU / L streptomycin) in an incubator at 37°C and 5% CO2.
[0041] 2. Hypoxia treatment conditions for BRL cells:
[0042] BRL cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. The hypoxic environment was 1% O2, 5% CO2, and 95% N2. The rest of the culture conditions were the same as normal cells. The hypoxic injury time was according to the experimental design requirements. conduct.
[0043] 3. Hypoxic injury experiment of BRL cells:
[0044] Three groups were designed in the experiment, namely control group, hypoxia treatment group 1 and hypoxia treatment group 2.
[0045] Digest BRL cells with 0.1% trypsin solution, make cell suspension, inoculate i...
Embodiment 2
[0054] Embodiment two, see appendix Figure 4-5 .
[0055] Determination of protein expression of hypoxia-inducible factor (HIF-1α) in rat liver cells (BRL)
[0056] 1. BRL cell culture conditions:
[0057] Described with specific embodiment 1.
[0058] 2. Hypoxia treatment conditions for BRL cells:
[0059] Described with specific embodiment 1.
[0060] 3. Hypoxic injury experiment of BRL cells:
[0061] Three groups were designed in the experiment, namely control group, hypoxia treatment group 1 and hypoxia treatment group 2.
[0062] Digest BRL cells with 0.1% trypsin solution, prepare cell suspension, inoculate in three six-well plates respectively, adjust the density to 1.0×106 / mL, add 3.0×105 cells per well, that is 33 microliters For the cell suspension, add 167 microliters of complete medium to 200 microliters, culture the cells for 24 hours, absorb the original medium after the cells are completely attached to the wall, and wash twice with PBS.
[0063] Control...
Embodiment 3
[0072] Embodiment three, see appendix Figure 6 .
[0073] Application of hypoxic injury model to screen and validate anti-hypoxic drugs
[0074] 1. BRL cell culture conditions:
[0075] Described with specific embodiment 1.
[0076] 2. Hypoxia treatment conditions for BRL cells:
[0077] Described with specific embodiment 1.
[0078] 3. BRL cell hypoxia model to verify the anti-hypoxic effect of acetazolamide:
[0079] Inoculate the BRL cell suspension in 96-well plates, adjust the density to 1.0×106 / mL, add 1.0×104 cells per well, that is, 10 microliters of cell suspension, add 90 microliters of complete medium to 100 microliters 1, culture the cells for 24 hours, after the cells are completely adhered to the wall, the original medium is sucked off, and the cells are washed twice with PBS.
[0080] Construct hypoxic injury cell model according to the method described in the present invention, promptly the cell adheres to the wall and grows on the 96-well plate, after w...
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