Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.

Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.     

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O

Summary Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.     

9-顺式维甲酸在原代培养猪前体脂肪细胞分化中的调控作用

采用RT-PCR、油红O染色法、油红O染色提取法和分光光度计法,探讨不同浓度9-顺式维甲酸(9-cisRA)(0-10$mol/L)对体外原代培养猪前体脂肪细胞分化的影响及其可能机制。结果表明,9-cisRA在脂肪细胞分化中因浓度不同而发挥不同作用。低浓度9-cisRA(0-10nmol/L)促进前体脂肪细胞分化,并上调RXRα、PPARγmRNA表达,增加前体脂肪细胞分化标志酶3-磷酸甘油脱氢酶(glyc-erol-3-phosphate dehydrogenase,GPDH)的活性;高浓度9-cisRA(100nmol/L-10$mol/L)则抑制前体脂肪细胞分化,并下调RXRα、PPARγmRNA表达,减少GPDH的活性。结果提示9-cisRA在猪前体脂肪细胞分化中,可能通过调控RXRα和PPARγ基因表达变化来发挥其促进或抑制作用。     

大鼠脂肪基质细胞成脂分化及慢病毒感染的实验研究

目的诱导大鼠脂肪基质细胞成脂分化,观察慢病毒感染效果。方法大鼠脂肪基质细胞培养至第3代后,间接免疫荧光法鉴定细胞表面抗原CD44,并采用MTT法绘制生长曲线;第3代基质细胞成脂诱导分化为脂肪细胞,油红O染色法鉴定,并行慢病毒感染。结果第3代脂肪基质细胞表面抗原CD44表达呈阳性;细胞生长曲线呈"S"形。细胞经成脂诱导分化剂诱导10d后,胞内有大量脂滴形成,脂滴大小不等。油红O染色显示脂滴被染成红色;携带GFP报告基因的慢病毒可感染成熟脂肪细胞,感染率约为80%,且细胞被感染后状态良好。结果 慢病毒可高效感染由大鼠脂肪基质细胞成脂诱导分化成的脂肪细胞,为研究脂肪细胞的基因功能、相关疾病的基因治疗提供了一个有效工具。     

The value of simple lipid stains for typing skeletal muscle fibres

Summary Skeletal muscle fibre types can be distinguished rapidly with simple lipid stains. Comparative studies showed that Sudan Black B is superior to Oil Red O for this purpose and that optimum staining is obtained using unfixed sections or sections fixed in calciumglutaraldehyde. Factors that possibly influence the staining reaction, such as freeze-thawing, are considered. The stained lipids were identified by thin layer chromatography.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.     

用成熟脂肪建立一种新的猪前体脂肪细胞培养模型

用去分化的成熟脂肪细胞建立一种新的具有再增殖和再分化能力的猪前体脂肪细胞模型. 用“天花板” 培养法分离、培养1～3日龄仔猪皮下成熟脂肪细胞, 显微镜下观察细胞形态变化并计数, 流式细胞术检测细胞周期;油红O染色法检测脂肪细胞分化率, RT-PCR分析前体脂肪细胞标志基因Pref-1及成熟脂肪细胞关键转录因子PPARγ和C/EBPα等mRNA表达情况. 发现刚贴壁的细胞为单室脂滴成熟脂肪细胞, 油红O染色完全阳性; 14d后这种成熟脂肪细胞完全去分化为无脂滴的纤维状细胞, 并表达前体脂肪细胞标志基因Pref-1, 油红O染色阴性. 这种去分化的前体脂肪细胞在成脂诱导剂作用下,可重新分化为成熟的脂肪细胞. 结果证实,成熟脂肪细胞去分化后的前体脂肪细胞可重新增殖、分化为成熟脂肪细胞, 是一种新的有效的前体脂肪细胞模型.     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.     

Histochemical and thin layer chromatographic analyses of neutral lipids in Leucochloridiomorpha constantiae (Trematoda) adults

Histochemical and thin layer Chromatographic studies were made on neutral lipids in adult Leucochloridiomorpha constantiae (Trematoda) obtained from the bursa of Fabricius of experimentally infected domestic chicks. Oil Red O staining of adults revealed neutral lipids in the suckers, parenchyma and eggs, and trace amounts in the cecal epithelium and lumina of the gut. TLC analysis detected triglycerides and free sterols as the major neutral lipid fractions of adults. Lesser amounts of free fatty acids, cholesterol esters, mono- and diglycerides were also found.     

Measurement of the specific lipid content of attached cells in microtiter cultures

A method has been described to quantify intracellular neutral lipid content in attached cells in microtiter cultures. The procedure was based on oil red O staining of neutral lipid and Coomassie brilliant blue G-250 staining of total cellular protein. Results were expressed as the ratio of lipid to protein, the "specific lipid content" index. This measurement was shown to closely correspond to actual lipid per cell measurements under experimental conditions. The procedure was specific for neutral lipids and sensitive (greater than or equal to 50 ng triglyceride/well). Additionally, cell proliferation measurements could be made simultaneously, using protein staining data. Chromatic endpoints were measured using a spectrophotometer capable of reading individual wells of a microtiter plate. The procedure is recommended for applications in which the endpoint is neutral lipid droplet accumulation in attached cultured cells.     

Accumulation and excretion of neutral lipids in the metacercaria of Leucochloridiomorpha constantiae (Trematoda) maintained in vitro.

Histochemical and thin-layer chromatographic analyses were made on neutral lipids in Leucochloridiomorpha constantiae (Trematoda) metacercariae incubated in non-nutrient Locke's solution at 37.5 or 42 C for 24 hr. As determined by Oil Red O staining, lipid droplets accumulate mainly in the intestine of incubated worms. Attempts to distinguish specific neutral lipid fraction of metacercariae obtained directly from snails is free sterol. During incubation, metacercariae accumulate triglycerides and to a lesser extent cholesterol esters, and excrete free sterol into the medium.     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.     

Vibrational spectroscopy-based quantification of liver steatosis

The liver plays a central role in lipid metabolism, and abnormal lipid accumulation in the liver is a key feature of Non-Alcoholic Fatty Liver Disease. In experimental studies, quantification of liver steatosis is commonly based on lipids staining or biochemical analysis. Here, we present a spectroscopic approach for quantitative analysis of the lipid content in the freeze-dried liver. The method is based on vibrational spectroscopy (Raman and infrared) measurements applied for Partial Least Squares (PLS) regression modeling. The obtained PLS models show a good correlation of the spectroscopic data with the reference histological evaluation of steatosis based on Oil Red O (ORO)-stained images of liver cross sections. Vibrational spectroscopy with PLS-based modeling described here represents a useful approach for the fast assessment of the liver steatosis in a small sample of freeze-dried liver tissue. In conclusion, our work demonstrates the easy-to-use method that can be applied in laboratory routine as a beneficial alternative to the established ORO staining.     

Network distribution of mitochondria and lipid droplets in human muscle fibres

The objective of the present study was to develop a combination of fluorescent stains that would allow visualisation of the network of mitochondria and lipid droplets (intramyocellular lipids or IMCL) in human skeletal muscle fibres by means of conventional and confocal microscopy. Muscle biopsies were taken from the vastus lateralis of three lean, healthy and physically active male subjects. Frozen muscle sections were stained for mitochondria using antibodies against three mitochondrial proteins; porin, cytochrome c oxidase (COX) and NADH-ubiquinol oxidoreductase and neutral lipids were stained with oil red O. Anti-COX staining produced images with the strongest fluorescence signal and the highest resolution of the mitochondrial network and this stain was successfully combined with the antibody against type I fibre myosin. A highly organised matrix arrangement of mitochondria within the sarcomeres (in pairs at the I-band) was observed in the oxidative type I fibres. The density of mitochondria was the highest in the subsarcolemmal region. Anti-COX staining was combined with oil red O demonstrating that in type I fibres lipid droplets are mainly located in the space between the mitochondria.     

Secreted adiponectin as a marker to evaluate in vitro the adipogenic differentiation of human mesenchymal stromal cells

Background aimsMultipotency is one of the hallmarks of mesenchymal stromal cells (MSCs). Given the widespread adoption of MSC-based clinical applications, the need for rapid and reliable methods to estimate MSC multipotency is demanding. Adipogenic potential is commonly evaluated by staining cell lipid droplets with oil red O. This cytochemical assay is performed at the terminal stage of adipogenic induction (21–28 days) and necessitates the destruction of the specimen. In this study, we investigated whether it is possible to assess MSC adipogenic differentiation in a more efficient, timely and non-destructive manner, while monitoring in vitro secretion of adiponectin, a hormone specifically secreted by adipose tissue.MethodsA commercially available enzyme-linked immunosorbent assay kit was used to quantify adiponectin secreted in the culture medium of adipo-induced human bone marrow–derived MSCs. Oil red O staining was used as a reference method.ResultsAdiponectin is detectable after 10 days of induction at a median concentration of 5.13 ng/mL. The secretion of adiponectin steadily increases as adipogenesis proceeds. Adiponectin is undetectable when adipogenic induction is pharmacologically blocked, inefficient or when human MSCs are induced to differentiate toward the osteogenic lineage, proving the specificity of the assay. Furthermore, the results of adiponectin secretion strongly correlate with oil red O quantification at the end of induction treatment.ConclusionsOur results demonstrate that quantification of secreted adiponectin can be used as a reliable and robust method to evaluate adipogenic potential without destroying samples. This method provides a useful tool for quality control in the laboratory and in clinical applications of human MSCs.     

Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch''s membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM–choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM–choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-μm-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM. (J Histochem Cytochem 57:731–739, 2009)