Diabetologie und Stoffwechsel 2016; 11 - P228
DOI: 10.1055/s-0036-1580975

Valid assessment of lipid content of cultured adipocytes using oil red O

NA Kraus 1, B Rudolphi 1, B Zapp 1, C Wanner 1, D Kraus 1
  • 1Universitätsklinikum Würzburg, Medizinische Klinik und Poliklinik 1, Würzburg, Germany

Background: Cell culture is an important tool to study adipocyte biology. Oil red O (ORO) staining is commonly used to assess total lipid content of cultured adipocytes, as a surrogate for the degree of differentiation. However, despite widespread use of this method, its validity to measure total lipid content in the linear range has never been confirmed and published.

Aim: To develop and validate a protocol that measures lipid content of cultured adipocytes in a linear range.

Method: Differentiated 3T3-L1 adipocytes were washed in phosphate-buffered saline, fixed in formalin, stained with ORO, and washed several times with distilled water. Dye was eluted with isopropanol, transferred to a microtiter plate, and adsorption was measured at 510nm. The amount of cells was varied in multiple steps by scraping off the culture dish; timing and concentration of the ORO solution and eluate volume were optimized in order to maximize the linear relationship between amount of cells and absorption.

Results: The cell-absorption relation was non-linear at high as well as low concentrations of ORO. Duration of staining affected linearity as well. Best correlation was achieved at 0.2% ORO for 30 min (R2= 0.982, p < 0.001).

Conclusion: None of the protocols in the published literature are optimized to measure in the linear range. When ORO staining is employed to compare the degree of differentiation between differently treated culture plates, our validated protocol should be used.