Quantitative assessment of adipocyte differentiation in cell culture

Abstract

Adipocyte cell culture is an important tool for mechanistic studies of energy metabolism. Many factors affect the differentiation of adipocytes in culture. Oil red O staining can be used to assess the degree of differentiation. However, the validity of this method for quantitative analysis has not yet been established. Here we show that a protocol with arbitrarily chosen parameters does not measure in the linear range and is not suitable for quantitative analysis (R 2 D 0.077, p D 0.382), and develop and validate an optimized protocol for quantitative oil red O staining of cultured adipocytes. 3T3-L1 preadipocytes and adipocytes are fixed with 4% formaldehyde and stained with 0.2% oil red O solution in 40% 2-propanol for 30 minutes. Dye is eluted with 2-propanol, and absorption of the eluate is measured photometrically at 510 nm. This optimized protocol achieves excellent correlation between defined amounts of differentiated adipocytes on constant-size culture plates and photometric absorption (R 2 D 0.972, p D 6.585E-14). The performance of the method is independent of the culture plates used. Thus, the optimized oil red O staining protocol can be universally employed to quantitatively assess adipocyte differentiation.

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis

“…For this reason, we characterised the formation of lipid vacuoles using Oil Red O (Kraus et al 2016) in 2D monolayer and 3D microtissue cultures. In 2D monolayer cultures, large lipid vacuoles stained with Oil Red O were observed in BMSC cultures derived from all three donors in adipogenic medium, with little or no staining observed in maintenance Fig.…”

Rab27a Regulates Human Perivascular Adipose Progenitor Cell Differentiation

“…Human PVAT-APC were induced to differentiate towards an adipogenic lineage for 7 days using previously established methods [30], and neutral lipid accumulation visualized by oil red O (ORO) staining [31]. Compared to non-induced PVAT-APC, induced cells showed accumulation of multilocular adipocytes (Fig.…”

Novel properties of myoferlin in glucose metabolism via pathways involving modulation of adipose functions

“…Lipid accumulation was visualized on day 10 by oil red O staining and quantitatively evaluated as previously described. 10 Isolation and culture of thioglycolate-elicited peritoneal macrophages were performed as previously described. 11 The acquired cells were cultured in DMEM with 10% FCS and stimulated with or without 100 ng/mL TNFα for 24 hours.…”