Method for preparing brown adipocyte
A brown fat and cell technology, applied in the field of brown fat cells and their preparation, can solve problems such as high cost, complicated induction technology, and cancerous cells
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Embodiment 1
[0206] Fibroblasts derived from normal human skin (human dermal fibroblasts; HDF) were suspended in a normal medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Convert it to 1×10 4 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. The next day, the culture supernatant was aspirated, and 500 μL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with a compound or the like was added as described in the figure.
[0207] Adipocyte induction medium is DMEM+MDI medium supplemented with 10% FBS (DMEM supplemented with 10% FBS supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 0.5 μm dexamethasone and 1 μg / mL insulin) .
[0208] The concentrations of the additives are as follows:
[0209] T3: 1nM
[0210] Rosiglitazone: 1 μm
[0211] D4476: 2μm
[0212] Pifithrin alpha [p53 inhibitor]: 5 μm
[0213] SB431542: 2μm
...
Embodiment 2
[0219] Fibroblasts derived from human normal skin (human dermal fibroblasts; HDFs) were suspended in a normal medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Convert it to 1×10 4 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. On the next day, the culture supernatant was aspirated, and 500 μL / well of normal medium, adipocyte induction medium, or adipocyte induction medium supplemented with various small molecular compounds or the like was added as described in the figure.
[0220] Adipocyte induction medium is DMEM+MDI medium supplemented with 10% FBS (DMEM supplemented with 10% FBS supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 0.5 μm dexamethasone and 1 μg / mL insulin) .
[0221] The concentrations of the additives are as follows:
[0222] T3: 1nM
[0223] Rosiglitazone: 1 μm
[0224] D4476: 2μm
[0225] Pifithrin alpha [p53 inhibitor]: 5 μm ...
Embodiment 3
[0233] Carry out the same experiment with embodiment 2, prepared the cell that cultured with common culture medium, in the adipocyte induction medium that has added T3 and rosiglitazone, cultivated the cell of 14 days, and added T3, Rogner Cells cultured for 14 days in adipocyte induction medium of Litazone and D4476. To these cells, 10 μM of isoproterenol or FSK was added as described in the figure. A group without addition was also produced as a control. After 5 hours, the culture solution was aspirated from each well, and after washing with PBS(-), total RNA was extracted from the cells using ISOGEN II. qRT-PCR was performed in the same manner as in Example 2. Quantification was performed by the ratio of the mRNA level of the UCP1 gene to the mRNA level of the β-actin gene, and the value of fibroblasts cultured in a normal medium was set to 1 for calculation.
[0234] show the result in image 3 . It can be seen that the cells cultured for 14 days in the adipocyte indu...
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