Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio (2023)


Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is an ideal probe for the detection of lipids as it exhibits high affinity, specificity and sensitivity to the degree of hydrophobicity of lipids. The latter feature results in a shift of the emission spectrum from red to yellow, in the presence of polar and non-polar lipids, respectively (Greenspan and Fowler, 1985). Accordingly, cytoplasmic membranes, which are composed mostly of phospholipids, are generally stained in red whereas lipid droplets, composed of esterified cholesterol and triglycerides (Bartz et al., 2007; for a review see Martin and Parton, 2006), are stained in yellow. For this property, since its discovery, Nile Red has been widely employed in studies concerning lipid storage diseases and lipogenesis (Fowler and Greenspan, 1985). However, a quantitative ratio of red and yellow emissions has been evaluated only in flow cytometry studies aiming to assess lipid changes in differentiating adipocytes (Smyth and Wharton, 1992), Niemann-Pick cells (Brown et al., 1992) and promonocytic leukemia cells (Vejux et al., 2005). On the other hand, flow cytometric analysis, which provide a single cumulative value for the whole cell, are substantially ambiguous about the nature and localization of intracellular lipids, as many different organelles (ER, Golgi, mitochondria, lipid droplets, etc.) produce a strong red emission. A better approach is allowed by image analysis by which subcellular structures can be recognized and evaluated separately. On the other hand, Nile Red ratiometric methods have not yet been applied in fluorescence microscopy studies. All previous microscopic investigations used Nile Red staining only for qualitative evaluations.

In this work, we describe a simple ratiometric method accompanied by data obtained from a set of reference lipids with different hydrophobic properties (free cholesterol, oleyl cholesteryl ester, trioleine, monooleine, oleic acid and phosphatidylcholine). We also show Nile Red red/yellow emission ratios found in lipid droplets of 3T3 fibroblasts and the relevant changes induced in the same cells by drugs interfering with the cholesterol cycle (Sandoz 58-035, U18666A and bafilomycin-A1).

Section snippets

Cells and treatments

Swiss 3T3 fibroblasts (ATCC collection, CCL-92) were grown in phenol red-free Dulbecco's modified Eagle's medium with 4500mg/L glucose, supplemented with 10% fetal bovine serum, in a 5% CO2 incubator at 37°C. Cells were seeded in number of 105cells/cm2 in 35mm glass-bottomed dishes (MatTek, Ashland, MA). One day after seeding cells were treated with 4μM Sandoz 58-035 for 18h, 4.34μM U18666A for 18h or 10nM bafilomycin-A1 for 60min. Cells were then fixed with 4% paraformaldehyde in PBS for 30min


Reference lipids (free cholesterol, oleyl cholesteryl ester, triolein, oleic acid, monoolein and phosphatidylcholine) stained with Nile Red showed a good linear relationship of red and green emissions (Fig. 1C). Red/yellow emission ratios ranged from 1.9 (cholesterol ester) to 82.1 (monoolein) and were highly discriminant for all lipids tested. Since lipid droplets accumulate different lipids, mostly represented by cholesterol esters and triglycerides, we also measured the red/yellow emission


Since its discovery, Nile Red has proven to be an excellent probe for lipids at histological and cytological level (Greenspan et al., 1985). Nile Red may be dissolved in acqueous media, thus avoiding the use of non-polar solvents which extract lipids. In water, Nile Red fluorescence is strongly quenched, so it can be maintained in the medium without appreciable background. Nile Red also stains living cells, being highly membrane-permeant and not acutely toxic at nanomolar concentrations. In

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  • MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies

    2023, Metabolism: Clinical and Experimental

    Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms.

    PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis.

    MLX knockdown in PHH altered lipid metabolism (decreased DNL (p<0.05), increased fatty acid oxidation and ketogenesis (p<0.05), and reduced lipid accumulation (p<0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p<0.001) and insulin-stimulated pAKT levels (p<0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p<0.05 and p<0.01, respectively) and TG (p<0.05 and p<0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p<0.01).

    We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

  • Hepatocyte mARC1 promotes fatty liver disease

    2023, JHEP Reports

    Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with invitro and invivo studies of mARC1 knockdown.

    Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an invitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)–siRNA to understand the invivo impacts of MTARC1.

    We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. Invitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and invivo GalNAc–siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown invitro and invivo.

    Collectively, our findings from human genetics, and invitro and invivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD.

    We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 forNAFLD.

  • Exposure to non-esterified fatty acids in vitro results in changes in the ovarian and follicular environment in cattle

    2022, Animal Reproduction Science

    Negative energy balance (NEB) in the postpartum period of dairy cows is associated with reduced fertility to insemination later in lactation. We hypothesized that elevated non-esterified fatty acids (NEFA) levels that occur during NEB result in accumulation of fatty acids within the ovarian tissue and preantral follicles, causing changes in ovarian gene expression that would indicate a response to injury. We performed ovarian cortex culture and oocyte maturation in medium containing a combination of palmitic, oleic and stearic acid (NEFA). Ovarian cortex was subjected to RNA sequencing and lipid content analysis via Nile Red staining and gas chromatography; oocytes were analyzed for maturation rate and mitochondrial mass and localization following in vitro maturation (IVM). Accumulation of lipids associated with the plasma membrane was increased in granulosa cells of preantral follicles exposed to NEFA in vitro; RNA sequencing revealed changes in biological functions associated with metabolic disease, stimulation of an inflammatory response, and reduction in glucose uptake. Oocyte maturation under high NEFA compromised nuclear, but not cytoplasmic maturation. These data demonstrate that exposure to NEFA in vitro affects the ovary, preantral follicles and cumulus-oocyte complexes, and provides further insight into the potential links between metabolic imbalance and infertility.

  • Nile Red staining for detecting microplastics in biota: Preliminary evidence

    2021, Marine Pollution Bulletin

    Nile Red is a lipophilic, metachromatic and solvatochromic dye used as an alternative or complementary method to aid identification of microplastics in routine analysis of biological samples. It was rarely used in biota since organic residues after the digestion step can be co-stained with possible overestimation of microplastics. The limits of using Nile Red in biota were investigated in marine mussels experimentally contaminated with low-density polyethylene (LDPE) microplastics. Stained particles were detected through magnified images obtained by stitching together thirty photographs of the filter surface of each sample. LDPE particles appeared yellowish and fluorescent and could be confused with certain organic residues. The smaller the fragments, the greater the difficulty in recognizing them. In particular, it was difficult to recognize LDPE particles based on their fluorescence if <180 μm in size. Regardless of the size, fluorescence of the items aids the operator in LDPE particles identification also in biota.

  • Comprehensive assessment of factors influencing Nile red staining: Eliciting solutions for efficient microplastics analysis

    2021, Marine Pollution Bulletin

    Monitoring microplastics in the environment based on the Nile red staining protocol has proven to be a newly emerged method in several instances. However, the methodology is still having the limitations of susceptibility, indiscrimination, and complexity, etc. The objectives of this paper are to explore the effects of wavelength, temperature, H2O2 and NaCl addition, plastic property, and fluorescent index on the Nile red staining in microplastics analysis and propose solutions to these inadequacies. Sample co-stained with H2O2final=10%) and NaCl (ωfinal=8.8%) will lower the fluorescence intensity of biogenic materials and reduce their interferences. Based on the fluorescence color and intensity of fused fluorographs, the combined fluorescent index for twelve microplastics was significantly different, thus could be preliminarily distinguished. An elevated staining temperature is propitious to fluorescent tagging with Nile Red. Finally, an improved protocol was proposed, which made the methodology streamlined in microplastics analysis.

  • Triclosan down-regulates fatty acid synthase through microRNAs in HepG2 cells

    2021, European Journal of Pharmacology

    Triclosan is a promising candidate of fatty acid synthase (FASN) inhibitor by blocking FASN activity, but its effect on FASN expression and the underling epigenetic mechanism remain elusive. In this study, the effect of triclosan on FASN mRNA and protein expressions in human HepG2 cells and the regulatory role of microRNAs (miRNAs) in the downregulation of FASN induced by triclosan were explored through experiments and bioinformatics analysis. The results showed that triclosan not only directly inhibited FASN activity, but also significantly decreased FASN mRNA and protein levels in human liver HepG2 cells. Nine miRNAs targeting FASN mRNA degradation were identified by miRNA prediction tools, and the expression levels of these nine miRNAs were then detected by real-time quantitative PCR. Triclosan significantly increased the expressions of the six miRNAs, namely miR-15a, miR-107, miR-195, miR-424, miR-497 and miR-503, leading to the downregulation of FASN. Further investigation revealed that the six triclosan-upregulated miRNAs played an important regulatory role in lipid metabolism and cell cycle by gene ontology annotations and pathway analysis. Consistent with the results of bioinformatics analyses, triclosan significantly reduced the intracellular lipid content by triglyceride assay, oil red O, BODIPY 493/503 and Nile Red staining, thereby inhibiting the growth of HepG2 cells through apoptosis. Taken together, our study reveals that triclosan downregulates FASN expression through a variety of miRNAs, providing new insight for triclosan as a FASN inhibitor candidate to regulate lipid metabolism in human hepatoma cells.

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Copyright © 2008 Elsevier Ltd. All rights reserved.


Why is Nile red hydrophobic? ›

Abstract. 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.

What is the excitation and emission of Nile red? ›

In triglycerides (a neutral lipid), Nile red has an excitation maximum of about 515 nm (green), and emission maximum of about 585 nm (yellow-orange). In contrast, in phospholipids (polar lipids), Nile red has an excitation maximum of about 554 nm (green), and an emission maximum of about 638 nm (red).

What lipids does Nile red stain? ›

Under these spectral conditions, nile red can be thought to be a general stain for lipids since we found that the dye can interact and fluoresce in the presence of phospholipid, cholesterol, choles- teryl esters, and triacylglycerols (Greenspan, P., and S. D.

What is the difference between oil red and Nile red? ›

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Is Nile red hydrophobic? ›

Nile red is a hydrophobic and metachromatic dye with poor solubility and fluorescence in water, with colour emission varying from deep red to strong yellow gold in hydrophobic environments. Depending on excitation and emission wavelength, the dye has been used to stain different hydrophobic molecules.

Is Nile red hydrophilic? ›

Nile red is an uncharged hydrophobic molecule whose fluorescence is strongly influenced by the polarity of its environment.

What is the emission spectrum of Nile Red? ›

Nile Red is a green crystalline powder with a molecular weight of 318.38. It is sometimes also known as Nile Blue A Oxazone. Nile Red is a fluorescent compound with an excitation peak at 559 nm and an emission peak at 635 nm, giving it a fairly large Stokes' Shift of 76nm.

What is the excitation emission wavelength of Nile Red in water? ›

Monitoring at 680 and 620 nm, the excitation maxima were found to be at 588 and 500 nm (with a hump at around 450 nm in the latter case), respectively.

What is the emission spectra of Nile Red? ›

Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm).

What is the principle of nile red staining? ›

Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution.

What does nile red stain do? ›

We report that the dye nile red, 9-diethylamino-5H- benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry.

What causes color change red for lipid? ›

Layer systems composed of polydiacetylene lipids are known to undergo an irreversible colorimetric transition from blue to red upon exposure to an external trigger (e.g., heat, pH change, specific ligand−target interaction).

Does nile red dissolve in water? ›

Nile Red (compound A) fluoresces at about 530 nm with good quantum yields in apolar solvents. In more polar ones its fluorescence emission shows a dramatic, and potentially useful, shift to about 640 nm, but its quantum yield is significantly reduced. Further, Nile Red has a very poor solubility in aqueous media.

What is the solvent for nile red? ›

Nile red is soluble in organic solvents such as DMSO and dimethyl formamide. The solubility of nile red in these solvents is approximately 1 mg/ml. Nile red is also slightly soluble in ethanol.

How do you use nile red dye to detect microplastics? ›

The standard procedure in the use of Nile-Red foresees the dissolution of the dye in acetone or methanol, followed by addition of few drops of the dye solution, to cover each filter containing microplastics. The analysis of the filters is performed after a contact time, ranging from 30 to 60 min (Rumin et al.

What does Nile blue stain for lipid? ›

Nile blue staining

Nile blue is used for histological staining of biological preparations. It highlights the distinction between neutral lipids (triglycerides, cholesteryl esters, steroids) which are stained pink and acids (fatty acids, chromolipids, phospholipids) which are stained blue.

What is Nile red staining of macrophages? ›

Nile red was initially defined as a highly specific vital stain, allowing the detection of intracellular LDs in cultured aortic smooth muscle cells and peritoneal macrophages, as it selectively dissolves in the lipids and fluoresces with no local interaction with the tissue constituent (Greenspan et al., 1985).

What staining is best for lipid detection? ›

Sudan Red dye is used to stain lipids.

Is Nile Blue soluble in water? ›

Solubility : Soluble in ethanol, and water (50 g/L) at 25° C. Melting Point : >300° C (dec.)

How are nile red and Nile Blue different? ›

Nile Red has a broad range of emission and can be excited using the 488 nm or 565 nm lasers. Nile Blue is a far-red dye and is excited at 625 nm. Typically, Nile Red fluorescent signal in lipid droplets is observed in the orange-red channels whereas Nile Blue fluorescence is detected in far red channels.

What causes red emission? ›

At optical wavelengths, the most important of these transitions corresponds to a wavelength of 656.3nm in the red end of the spectrum. This is the wavelength of Hα, and it is this transition that gives emission nebulae their distinctive red colour.

What is the quantum yield of Nile Red? ›

The fluorescence emission spectrum of Nile Red dissolved in Dioxane. The excitation wavelength was 460nm. The quantum yield of this molecule is 0.7 (Sackett, 1987).

What are the colors of the three most intense lines in the emission spectrum of hydrogen? ›

(b) When the light emitted by a sample of excited hydrogen atoms is split into its component wavelengths by a prism, four characteristic violet, blue, green, and red emission lines can be observed, the most intense of which is at 656 nm.

What is the excitation wavelength of yellow? ›

Nuclear Yellow is a fluorescent compound with an excitation peak at 372 nm and an emission peak at 504 nm, giving it a fairly large Stokes' Shift of 132nm.

Which is greater the wavelength for excitation or for emission for fluorescence? ›

The emitted light is always of longer wavelength than the excitation light (Stokes Law) and continues so long as the excitation illumination bathes the fluorescent specimen.

What wavelength is red light excitation? ›

The resulting fluorescence is most intense in the red portion of the light spectrum, with a peak at around 635 nm and extending to about 720 nm.

What is the excitation emission wavelength of Nile Blue? ›

Nile Blue is a fluorescent compound with an excitation peak at 631 nm and an emission peak at 660 nm. It can be excited using a 633 nm laser paired with a 660/20 nm bandpass filter, a configuration that can be found, for example, in the BD FACSAria™ III.

What are wavelengths of emission? ›

The emission wavelength is the WaveLength of the radiation emitted by the object being measured, coming from the fluorescent dyes. The fluorescent emission is caused by a radiation source with a given Excitation Wavelength.

What are the three types of emission spectra? ›

Types of Spectra: Continuous, Emission, and Absorption.

Is Nile red soluble in water? ›

Nile Red (compound A) fluoresces at about 530 nm with good quantum yields in apolar solvents. In more polar ones its fluorescence emission shows a dramatic, and potentially useful, shift to about 640 nm, but its quantum yield is significantly reduced. Further, Nile Red has a very poor solubility in aqueous media.

What is the solubility of Nile Red? ›

Nile red is soluble in organic solvents such as DMSO and dimethyl formamide. The solubility of nile red in these solvents is approximately 1 mg/ml. Nile red is also slightly soluble in ethanol. Nile red is sparingly soluble in aqueous buffers.

What are the properties of Nile red? ›

Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution.

How do you dissolve Nile red in water? ›

Nile red is relatively insoluble in aqueous solutions and is first dissolved in acetone, then mixed rapidly with water immediately prior to application to the gel.

Does Nile Red stain phospholipids? ›

These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.

What is Nile Red for microplastics? ›

Nile Red (NR) is a hydrophobic, metachromatic, and photochemically stable dye commonly used in microplastics research. NR is an uncharged, organic, and heterocyclic compound composed of 5H-benzo[a]phenoxazin-5-one with a diethylamino group substituted at position 9 (Greenspan et al., 1985).

Which dye is most soluble in water? ›

Cationic dyes are most soluble in water as chloride, sulfate or nitrate salts. There are used as solvent dyes in free base form. Phosphotungstomolybdic acid salt is often used to obtain pigments. Furthermore, anionic dyes are frequently overdyed with cationic dyes to form insoluble double dye salts.

What is the molarity of Nile Red? ›

The molar extinction coefficient (ε) for Nile Red is 38000 cm-1M-1 .

Is Nile Red chemical? ›

Nile red is an organic heterotetracyclic compound that is 5H-benzo[a]phenoxazin-5-one substituted at position 9 by a diethylamino group. It has a role as a fluorochrome and a histological dye. It is an organic heterotetracyclic compound, a cyclic ketone, an aromatic amine and a tertiary amino compound.

Which is a unique feature of the Nile river? ›

The banks of the Nile all along its vast length contain rich soil as well, thanks to annual flooding that deposits silt. From space, the contrast between the Nile's lush green river banks and the barren desert through which it flows is obvious.

How do you prepare Nile Red solution for microplastics? ›

The standard procedure in the use of Nile-Red foresees the dissolution of the dye in acetone or methanol, followed by addition of few drops of the dye solution, to cover each filter containing microplastics. The analysis of the filters is performed after a contact time, ranging from 30 to 60 min (Rumin et al.

What is Nile Red stain used for? ›

Nile red is used to localize and quantitate lipids, particularly neutral lipid droplets within cells.


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