Adipogenic Differentiation Small Molecules Kit-1(Powder，With Oil Red O And Indomethacin) KLIN-1

Basic operations ( for reference only )

1. Inoculate stem cells: Take cells in the logarithmic growth phase, inoculate them into coated culture vessels at a cell density of 2×10 ⁴ cells/cm ^{2 ,}

 ^{and culture them to a confluence of 90-100% at 37°C in a 5% CO} ₂ culture environment. Discard the supernatant and add adipogenic differentiation medium

 induction medium (containing dexamethasone, insulin, IBMX, and indomethacin/rosiglitazone).

2. Cell differentiation induction: Cultivate in a culture environment of 37°C and 5% CO ₂ for about 2-3 days,

 then replace with adipogenesis induction differentiation medium maintenance medium (containing only insulin) and culture for 1 day.

3. Continue induction according to the above frequency of medium replacement (usually 3 cycles, about 14 days), 

and pay attention to the changes in cell morphology. Based on the number and size of lipid droplets formed by cell induction,

the time to terminate cell induction is determined and stained for identification.

4. Cell fixation: Aspirate the culture medium and wash once with an appropriate amount of 1×PBS. After discarding,

 cover the bottom of the culture vessel with an appropriate amount of 4% neutral formaldehyde solution and fix at room temperature for 30-60 minutes.

 Discard the fixative and wash twice with 1×PBS.

5. Oil Red O staining: Add an appropriate amount of working solution and let stand for 30 minutes; absorb the Oil Red O staining solution, wash twice with 1×PBS,

 and add an appropriate amount of 1×PBS to prevent the cells from drying out.

6. Induction evaluation: Observe the lipogenic staining effect under a microscope, and perform image collection and induction evaluation; when induction is successful, 

the lipid droplets will combine with Oil Red O dye and appear red or orange.