Adipocyte Differentiation Assay

Adipocyte Differentiation Assay

1.  Plate cells in culture dish (1 x 105/ 6 well plate for stromal cells).  Need two wells per condition for staining, more if you want to collect RNA.

2.  Allow cells to grow to confluence.

3.  Once cells have been at confluence for 2 days begin adding differentiation media-regular growth media plus:

0.1µm Dexamethasone

0.5mM IBMX

0.5µg/mL human insulin

4.  Add differentiation media to one well and regular growth media to other well so that you have an undifferentiated control well of cells.

5.  Refeed every 2-3 days, making fresh differentiation media right before adding.

6.  Lipid accumulation should begin around 10 days but this is just an average

7.  Stain cells treated with control and differentiation media with Oil Red O on day 21.

8.  Image cells

9.  Collect RNA if you set up enough wells to do so.  Check changes in gene expression:

PPAR gamma (should increase)

FABP4 (should increase)

Klf2 (should decrease)

10.  Continue with quantification of Oil Red O if you want.