Adipocyte Differentiation Assay

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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.

Adipocyte Differentiation Stain (Oil Red O)

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Oil Red O staining

  1. Rinse cells two times with PBS
  2. Fix cells in 1mL/6-well 10% formalin/PBS for one hour (no more than 2 hours) at RT.
  3. Rinse plates two times with ddH2O
  4. Stain with 1mL/6-well Oil Red O (stain prep below) for 2 hours at RT.
  5. Aspirate stain and wash cells gently with water a many times (around 15-20, until background stain comes off).
  6. Stained fat droplets will be dark brown/red in color.
  7. Leave some ddH2O on the plates and image.
  8. Either keep moist with PBS or air dry.

Preparation of Oil Red O:

  1. 1.05g Oil Red O dissolved in 300mL isopropanol and leave overnight at RT w/o stirring
  2. Filter with 0.45um membrane, add 250mL ddH2O and leave solution overnight at 4°C without stirring
  3. Filter 1x with 0.45um filter
  4. Filter 1x with 0.22um filter
  5. store at RT

Oil Red O quantification

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  1. Remove all water after imaging cells.
  2. Place 6 well dishes (or other culture vessel) into 37°C incubator for 10-15 minutes.
  3. Wait until well is completely dry (no water left at all!)
  4. Add 300uL 100% isopropanol/6 well plate and wash stain off the cells, collecting from the top of the well to the bottom.
  5. Transfer isopropanol  to 96 well plate
  6. Read absorbance of 96 well plate at 510nm.
  7. To calculate the stain compare the differentiated absorbance to the undifferentiated absorbance (i.e. Abs 510nm diff/Abs 510nm undiff).

Osteoblast Differentiation Assay

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Osteoblast 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:
    0.01M β-glycerol phosphate
    100mM Ascorbic Acid
  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. Mineralization should occur around 17-19 days
  7. Stain cells treated with control and differentiation media with Alizarin Red S on day 21.
  8. Image cells
  9. Collect RNA if you set up enough wells to do so.  Check changes in gene expression (these should all increase if differentiation occurred):

AP

RunX2

Osterix

Osteocalcin

Osteoblast Differentiation Stain (Alizarin Red S)

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Alizarin Rd Staining

  1. Rinse cells very gently one time with water and then very gently add 2% Alizarin Red S stain (preparation below) for 5-10 min at RT
  2. Aspirate stain and wash cells very gently several times with water until wash is pretty clear (can be a very light red).
  3. Add PBS to the well to fix the stain and then aspirate off relatively quickly.  Allow well to air dry completely.
  4. Take phase images.

Preparation of Alizarin Red S

  1. dissolve 2g Alizarin Red S in 100mL dH2O and adjust to pH 4.1-4.3 with ammonium hydroxide
  2. stain can be stored at RT.