Nile red staining

Manufactured by Merck Group

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Nile Red is a fluorescent dye commonly used in biological and chemical research. It is a lipophilic stain that selectively binds to neutral lipids, allowing for the visualization and quantification of lipid droplets within cells or tissue samples. The core function of Nile Red staining is to provide a reliable method for the detection and analysis of intracellular lipid accumulation.

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10 protocols using nile red staining

Microalgal Lipid Quantification and Fatty Acid Analysis

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Cellular neutral lipid content was determined by using Nile Red staining (Sigma, USA) according to Yang et al. [11 (link)]. Staining was performed in a 96-well microtitre plate in triplicates and fluorescence was recorded using Synergy 4 Hybrid (BioTek, USA) at a wavelength of 530 nm excitation and 580 nm emission. For confocal microscopic observation of microalgal morphology, cells were stained with Nile red and lucifugally incubated for 10 min at room temperature. Stained cells were observed under a laser-scanning confocal microscope Zeiss LSM510meta (Zeiss, Germany) with an excitation wavelength of 488 nm and an emission wavelength of 505–550 nm.
Total lipids from the microalgae were extracted as per the protocol reported by Bligh and Dyer [27 (link)]. Cells were subjected to nitrogen deprivation to validate the lipid accumulating capability as previously described [9 (link)]. Fatty acid composition of the total lipids was analyzed as fatty acid methyl esters (FAMEs) by using gas chromatography–mass spectrometry (GC–MS) according to the protocol reported previously [11 (link)].

Zou L.G., Chen J.W., Zheng D.L., Balamurugan S., Li D.W., Yang W.D., Liu J.S, & Li H.Y. (2018). High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum. Microbial Cell Factories, 17, 54.

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Quantifying Intracellular Lipid Accumulation

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Intracellular lipid accumulation was quantified using Nile Red staining (Sigma Chemical) according to the manufacturer’s instructions. Briefly, cultures were fixed with 4% formaldehyde in PBS for 20 min at room temperature, washed, treated with 1 μM Nile Red and counterstained with Hoechst 33258 (Sigma Chemical). Images were taken using a Carl Zeiss LSM700 confocal microscope and analyzed using ImageJ software. Lipid accumulation is presented as lipid intensity normalized to Hoechst. A minimum of four regions were quantified for each experimental condition.

Levy G., Habib N., Guzzardi M.A., Kitsberg D., Bomze D., Ezra E., Uygun B.E., Uygun K., Trippler M., Schlaak J.F., Shibolet O., Sklan E.H., Cohen M., Timm J., Friedman N, & Nahmias Y. (2016). Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection. Nature chemical biology, 12(12), 1037-1045.

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Nile Red Adipocyte Staining Protocol

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Nile red staining (19123, Sigma) solution was prepared by dissolving Nile red in acetone at a concentration of 0.1 mg/mL. The solution was diluted with PBS at a ratio of 1:200 and added to induced adipocytes. The stained adipocytes were then observed under a fluorescence microscope.

Wu M., Qin M, & Wang X. (2023). Therapeutic effects of isoquercetin on ovariectomy-induced osteoporosis in mice. Natural Products and Bioprospecting, 13(1), 20.

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Oleate-induced lipid accumulation in HepG2 cells

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HepG2 cells were obtained from Dr Xin Chen's laboratory at the University of California, San Francisco. Sodium oleate was obtained from Sigma-Aldrich and was dissolved in Dulbecco's modified Eagle medium (DMEM) with 1% fatty acid free bovine serum albumin (BSA) (Sigma). Oleate treatment of HepG2 cells was carried out as previously described with minor revision.10 (link)
20 (link) Specifically, HepG2 cells were plated in four-well chamber slides with DMEM medium supplemented with 10% fetal bovine serum (Invitrogen). After 24 h, HepG2 cells were treated with either control medium (DMEM supplemented with 1% fatty acid free BSA) or medium containing oleate (0.5 mM). The cells were cultured for another 24 h, then lipid accumulation and miR-21 expression were determined by Nile Red Staining (Sigma-Aldrich) and qRT-PCR, respectively (see online supplementary materials and methods for details).

Wu H., Ng R., Chen X., Steer C.J, & Song G. (2015). MicroRNA-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the HBP1-p53-Srebp1c pathway. Gut, 65(11), 1850-1860.

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Multilineage Differentiation of AD-MSCs

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The differentiation capacity of adipose-tissue-derived mesenchymal stem cells was validated by differentiation into adipocyte, chondrocyte and osteocyte lines. They were seeded in a 24-well plate, 5 × 10⁴ cells/well, and after 24 h, the medium was replaced with a differentiation medium. For this purpose, commercially available Gibco’s StemPro^® Adipogenesis, Osteogenesis and cholndrogenesis differentiation kits were applied according to the manufacturer’s guidelines (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 21 days of upkeep, cells were fixed with 4% methanol-free formaldehyde (Molar Chemicals, Hungary) for 20 min at RT. The differentiation statuses of AD-MSCs were validated using different staining. For visualization of lipid-laden particles, Nile red staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was applied, Alizarin red staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was utilized to show mineral deposits during osteogenesis, and Toluidine blue staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was used to label the chondrogenic mass.

Szűcs D., Miklós V., Monostori T., Guba M., Kun-Varga A., Póliska S., Kis E., Bende B., Kemény L, & Veréb Z. (2023). Effect of Inflammatory Microenvironment on the Regenerative Capacity of Adipose-Derived Mesenchymal Stem Cells. Cells, 12(15), 1966.

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Adipogenic Differentiation of BMSCs

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BMSCs were cultured with complete DMEM-HG (High Glucose) supplemented with 10 μM dexamethasone, 50 μM indomethacin, 10 mM 3-isobutyl-1-methylxanthine, and 0.1 μM insulin (all from Sigma-Aldrich, St. Louis, MI, USA). Adipogenic differentiation was demonstrated by performing Nile Red staining (Sigma-Aldrich) to visualize lipid droplet formation. Quantification was achieved by measurement of Nile Red fluorescence (Ex/Em 485/540), which was normalized to the cell number quantified by staining with Hoechst 33258 dye (Life Technologies) and consecutive readout on a multimode microplate reader (TECAN M200 PRO) (67 (link)).

Andrzejewska A., Catar R., Schoon J., Qazi T.H., Sass F.A., Jacobi D., Blankenstein A., Reinke S., Krüger D., Streitz M., Schlickeiser S., Richter S., Souidi N., Beez C., Kamhieh-Milz J., Krüger U., Zemojtel T., Jürchott K., Strunk D., Reinke P., Duda G., Moll G, & Geissler S. (2019). Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties. Frontiers in Immunology, 10, 2474.

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Oleate-Induced Lipid Accumulation in HepG2 Cells

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HepG2 cells were obtained from Dr Xin Chen’s laboratory at the University of California, San Francisco. Sodium oleate was obtained from Sigma-Aldrich and was dissolved in Dulbecco’s modified Eagle medium (DMEM) with 1% fatty acid free bovine serum albumin (BSA) (Sigma). Oleate treatment of HepG2 cells was carried out as previously described with minor revision.^{10 (link)20 (link)} Specifically, HepG2 cells were plated in four-well chamber slides with DMEM medium supplemented with 10% fetal bovine serum (Invitrogen). After 24 h, HepG2 cells were treated with either control medium (DMEM supplemented with 1% fatty acid free BSA) or medium containing oleate (0.5 mM). The cells were cultured for another 24 h, then lipid accumulation and miR-21 expression were determined by Nile Red Staining (Sigma-Aldrich) and qRT-PCR, respectively (see online supplementary materials and methods for details).

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Lipid Droplet and Cholesterol Staining

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Nile Red staining (Sigma) was performed in solution on paraformaldehyde fixed iSREs, at a concentration of 2.5ug/ml for 30 min at room temperature. Filipin staining (Sigma) also occurred in paraformaldehyde fixed cells at a concentration of 50ug/ml for 2 hr at room temperature after cells were pre-blocked in 1.5mg/ml glycine in PBS. All staining was performed in the dark, and cells were then cytospun on slides, counterstained with DAPI (2ug/ml, Sigma), mounted, and viewed with widefield fluorescence microscopy.

McGrath K.E., Koniski A.D., Murphy K., Getman M., An H.H., Schulz V.P., Kim A.R., Zhang B., Schofield T.L., Papoin J., Blanc L., Kingsley P.D., Westhoff C.M., Gallagher P.G., Chou S.T., Steiner L.A, & Palis J. (2024). BMI1 regulates human erythroid self-renewal through both gene repression and gene activation. bioRxiv.

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Intracellular Lipid Quantification with Nile Red

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To examine intracellular lipid accumulation, AML-12 cells were incubated with Nile red staining (Sigma, Missouri, United States). The images were observed with a fluorescence microscope.

Chu J., Yan R., Wang S., Li G., Kang X., Hu Y., Lin M., Shan W., Zhao Y., Wang Z., Sun R., Yao J, & Zhang N. (2021). Sinapic Acid Reduces Oxidative Stress and Pyroptosis via Inhibition of BRD4 in Alcoholic Liver Disease. Frontiers in Pharmacology, 12, 668708.

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Newborn Gammarid Lipid Droplet Visualization

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Newborn gammarids freshly extracted from the female brood pouch were gently dried on a paper, pooled in groups of five individuals, and weighed using an ultra-microbalance (Sartorius Ultra Micro Balance MSU2.7S000DM, readability 0.1 μg, repeatability ± 0.25 μg).
Lipid droplets in newborn individuals were visualized using a stock solution of Nile red staining (technical grade, Sigma-Aldrich) prepared with 10 mg of Nile red to 100 mL of acetone (for HPLC, Carbo-Elba). Just before use, the working solution was prepared by diluting the stock solution to 2 mg L^-1 in synthetic water. Live individuals were then exposed to the Nile red working solution in the dark for 3 h at 14°C. Stained individuals were immobilized between the microscope slide and the cover slide, and images of the entire animal were taken at 5 × magnification for visualization of lipid droplets. Fluorescence images were obtained using a Leica DM 2500 with a L5 filter cuber (EX 480/40, EM 527/30) [28 (link)]. Twenty individuals per condition were analyzed.

Arambourou H., Fuertes I., Vulliet E., Daniele G., Noury P., Delorme N., Abbaci K, & Barata C. (2018). Fenoxycarb exposure disrupted the reproductive success of the amphipod Gammarus fossarum with limited effects on the lipid profile. PLoS ONE, 13(4), e0196461.

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