Changes

'''Materials'''

•DPBS – Lonza catalog # 17-512 or equivalent

•10% formalin, neutral buffered – VWR catalog # VW3239-4 or equivalent

•Oil red o – Sigma catalog # 0-0625 or equivalent • 99% isopropanol – Sigma catalog # 1-0398 or equivalent

•60% isopropanol

•Induced adipogenic cultures

•Conical filter paper – Whatman No. 1 or equivalent

'''Procedure'''

'''Fixing adipogenic cultures'''

•Remove cells from incubator and place in the fume hood. All procedures involving formalin must be performed in a fume hood.

'''Preparing oil red o stain'''

•Prepare the stock solution by weighing out 300 mg of oil red o powder and adding this to 100 ml of 99% isopropanol. This solution is stable for one year from the date on which it is prepared.

'''Staining adipogenic cultures'''

•Slightly tilting the plate, remove the formalin from the sides of each well with a pipettor and discard the formalin into a designated formalin waste receptacle. Remove the formalin from the control wells first.

•Keep the plates wet with water until they are ready to be viewed. • View the plates on a phase contrast microscope. Lipids will appear red and the nuclei will appear blue.

References:

Pittenger MF, Mackay AM, Bech SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147

Novikoff AB, Novikoff PM, Rosen OM, Rubin CS (1980) J. Cell Biol. 87:180-196