Bodipy 493 503

Manufactured by Cayman Chemical

Sourced in United States

BODIPY 493/503 is a fluorescent dye that emits green light when excited. It has an excitation maximum at 493 nm and an emission maximum at 503 nm. The dye can be used for various applications in cell biology and biochemistry.

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Lab products found in correlation

12 protocols using bodipy 493 503

Oil Red O and BODIPY Lipid Staining

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For Oil Red O staining, Oil Red O Stain Kit (ab150678, Abcam) was used. We incubated 6-μm fixed frozen tissue sections (formalin-fixed paraffin-embedded sections are not recommended) or fixed cells on a slide with Oil Red O Solution overnight at room temperature and followed the other procedures with the manufacturer’s instructions. Images were captured with a Zeiss Axio Scan Z1 light microscopy and examined in a blinded fashion.
BODIPY 493/503 (25892, Cayman Chemical) was used to label cellular lipid contents. Briefly, 6-μm fixed frozen tissue sections or fixed cells on a slide were incubated with 7.5–15 μM BODIPY 493/503 for 15 minutes at room temperature and washed with PBS. The sample was counterstained with DAPI and then processed for confocal imaging.

Li H., Dixon E.E., Wu H, & Humphreys B.D. (2022). Comprehensive single-cell transcriptional profiling defines shared and unique epithelial injury responses during kidney fibrosis. Cell metabolism, 34(12), 1977-1998.e9.

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Quantifying Cholesterol Uptake in SMCs

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Oil Red O staining was used to quantify the amount of cholesterol in the SMCs. Briefly, after cholesterol loading, SMCs were fixed in 4% (w/v) paraformaldehyde (PFA) and incubated in 60% isopropanol for 3 min. Then, cells were stained with Oil Red O and Harris' hematoxylin. Images were captured by a light-microscope (DMI3000B, Leica, Germany). The cellular cholesterol was extract by isopropanol and OD value at 520 nm was determined to represent the cholesterol concentration.
The cholesterol uptake by SMCs was identified by BODIPY 493/503 staining (Qiu and Simon, 2016). After 20 μg/ml cholesterol treatment for 72 h, SMCs were washed 3 times with Dulbecco's phosphate buffered saline (DPBS) and 2 μM BODIPY 493/503 was added (Cayman, USA) for 15 min at 37°C in dark. Then, cells were fixed in 4% (w/v) PFA for 15 min, and incubated with DAPI for 5 min to stain the nucleus. Images were captured by a laser scanning confocal microscope (Leica, TCS SP5 II, Germany). Cholesterol uptake was quantified by relative fluorescence intensity (FI).

Mao X., Tan Y., Wang H., Li S, & Zhou Y. (2021). Substrate Stiffness Regulates Cholesterol Efflux in Smooth Muscle Cells. Frontiers in Cell and Developmental Biology, 9, 648715.

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Quantification of Neutral Lipids in Treated BUVEC

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BUVEC (n = 5) were seeded into 25 cm² flasks (Sarstedt) and cultured until confluence. Then, the cells were treated with BLT-1 (2 µM) for 48 h. To determine if inhibitor treatments exerted an effect on neutral lipids, pre-treated cells were stained with BODIPY 493/503 (2 µg/mL, 1 h, 37 °C, 5% CO₂, Cayman Chemical). Afterwards, cells were washed twice in 1× PBS (600× g; 5 min) and samples were analyzed with a BD Accuri C6^® FACS cell analyzer (Becton-Dickinson, Heidelberg, Germany). The cells were gated according to their size and granularity, while BODIPY 493/503-derived signals were assessed in the FL-1 channel as described elsewhere [15 (link)].

Larrazabal C., López-Osorio S., Velásquez Z.D., Hermosilla C., Taubert A, & Silva L.M. (2021). Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI). Microorganisms, 9(11), 2372.

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Immunostaining and Lipid Droplet Visualization

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Cells were washed twice with PBS and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) in PBS for 10 min at room temperature. Afterwards, cells were washed three times and blocked/permeabilized in PBS containing 0.1% saponin (Sigma-Aldrich) and 3% normal donkey serum (NDS; Thermo Fisher Scientific) for 1 h at room temperature. Primary antibody incubation was performed at 4 °C overnight in PBS containing 0.1% saponin and 0.3% NDS. Afterwards, cells were washed three times with PBS containing 0.1% saponin and incubated for 1 h in PBS containing 0.1% saponin and 0.3% NDS with the respective secondary antibody. For triglyceride staining, permeabilized cells were incubated for 1 h with 2.5 μM BODIPY 493/503 (Cayman Chemicals) in PBS containing 0.1% saponin. Nuclei were stained with DAPI (Thermo Fisher Scientific). Cells were mounted in Fluoromount-G (Southern Biotech) before analysis. Images were acquired using an IX-71 microscope (Olympus). 4 random regions were analyzed per sample for quantification. Primary and secondary antibodies are listed in Supplementary Table 2.

Groeger M., Matsuo K., Heidary Arash E., Pereira A., Le Guillou D., Pino C., Telles-Silva K.A., Maher J.J., Hsiao E.C, & Willenbring H. (2023). Modeling and therapeutic targeting of inflammation-induced hepatic insulin resistance using human iPSC-derived hepatocytes and macrophages. Nature Communications, 14, 3902.

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Mitochondrial Morphology Analysis

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Cells were seeded on coverslips and treated with 5 μM Manz A or DMSO for 24 h. Cells were stained with 100 nM of MitoTracker™ Red CMXRos (Thermo Fisher Scientific) for 45 min in the dark. Following three washes of PBS, cells were fixed with 4% paraformaldehyde/PBS (w/v) for 15 min and stained with Bodipy 493/503 (Cayman Chemical) for 30 min at RT in the dark. EverBrite™ Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Biotium, Fremont, CA, USA) was used to immobilize the coverslips. Coverslips were sealed with nail polish. Fluorescent images were captured using a Leica DM IL inverted fluorescence microscope.

Lin L.C., Chang H.Y., Kuo T.T., Chen H.Y., Liu W.S., Lo Y.J., Hsia S.M, & Huang T.C. (2023). Oxidative stress mediates the inhibitory effects of Manzamine A on uterine leiomyoma cell proliferation and extracellular matrix deposition via SOAT inhibition. Redox Biology, 66, 102861.

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Multimodal Imaging of Cellular Structures

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Staining with non-antibody probes was performed following the manufacturer’s guidelines. ActinGreen ReadyProbes (Cat#R37110; Invitrogen) was used to visualize morphology, FLUO4-AM (Cat# F14201; Invitrogen) for microglia calcium imaging, BODIPY 493/503 (Cat#25892; Cayman Chemical) for lipid droplet staining, Isolectin GS-IB4 Alexa Fluor 594(Cat# I21413; Invitrogen) to label microglia before co-culture and Vybrant Alexa Fluor 594 Lipid Raft Labeling Kit (Cat# V34405; Invitrogen) for lipid raft staining. Microscopy was performed using a Zeiss LSM880 confocal system, and fluorescent Z stack images were quantified using IMARIS (Oxford Instruments).

Victor M.B., Leary N., Luna X., Meharena H.S., Scannail A.N., Bozzelli P.L., Samaan G., Murdock M.H., von Maydell D., Effenberger A.H., Cerit O., Wen H.L., Liu L., Welch G., Bonner M, & Tsai L.H. (2022). Lipid accumulation induced by APOE4 impairs microglial surveillance of neuronal-network activity. Cell stem cell, 29(8), 1197-1212.e8.

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Quantifying Lipid Accumulation in Cells

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Accumulation of lipids after 6 days of differentiation was measured by BODIPY staining in live cells. Media were replaced with 500 μl DMEM containing 10% FBS and 1% penicillin and streptomycin. BODIPY 493/503 (Cayman) was prepared to a working concentration of 1:500 in DMEM without serum or antibiotics. About 500 μl of this solution was added to the cells and incubated for 30 min at 37 °C. Cells were imaged using a fluorescence microscope (Evos M5000; Life Technologies).

Shinde A.B., Nunn E.R., Wilson G.A., Chvasta M.T., Pinette J.A., Myers J.W., Peck S.H., Spinelli J.B, & Zaganjor E. (2023). Inhibition of nucleotide biosynthesis disrupts lipid accumulation and adipogenesis. The Journal of Biological Chemistry, 299(5), 104635.

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Cellular Metabolic Assay Protocol

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AGS (1 × 10⁶) cells were seeded in 12-well plates. After treatment, the cells were incubated with Glutamate Colorimetric Assay Kit (Cayman, Ann Arbor, MI, USA), 2-NBDG (a fluorescent glucose kit; Cayman), or BODIPY™ 493/503 (a fluorescent lipid kit; Cayman), respectively, in the dark. The relative levels of glutamate, glucose uptake, and lipid accumulation were measured using a TECAN ELISA reader (Männedorf, Switzerland) at 450 nm and FACSCalibur™ flow cytometer at Ex/Em = 485/535 nm and 493/503 nm, respectively.

Lo Y.L., Wang T.Y., Chen C.J., Chang Y.H, & Lin A.M. (2022). Two-in-One Nanoparticle Formulation to Deliver a Tyrosine Kinase Inhibitor and microRNA for Targeting Metabolic Reprogramming and Mitochondrial Dysfunction in Gastric Cancer. Pharmaceutics, 14(9), 1759.

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Sheep Muscle Stem Cell Imaging

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Sheep MuSCs were cultured to 50% confluence and stabilized with 4% paraformaldehyde. Then, cells were treated with 0.5% Triton X-100 for 10 min, followed by incubation with phalloidin (4 U/mL, Solarbio Life Sciences, Beijing) for 20 min in the dark. Next, cells were stained by 5 µM BODIPY 493/503 (Cayman Chemical, Michigan) at 37 °C in the dark for 15 min. Finally, the nuclei were stained with DAPI, and anti-fluorescence attenuation sealing agent was used for sealing. The images were captured by super-resolution laser confocal microscopy (Nikon Corporation, Japan).

Chen M., Li Y., Xu X., Wang S., Liu Z., Qi S., Si D., Man Z., Deng S., Liu G., Zhao Y., Yu K, & Lian Z. (2024). Metabolic differences in MSTN and FGF5 dual-gene edited sheep muscle cells during myogenesis. BMC genomics, 25(1).

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Visualizing Lipid Droplets in Cells

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Lipid droplets were stained with BODIPY 493/503 (Cayman, USA). Fixed cells were incubated for 30 min at 37 °C in the dark with 0.2 g/mL BODIPY 493/503 in solution. Then, the cells were washed and counterstained with DAPI. The samples were visualized using a confocal microscope (Zeiss, LSM 800, Germany). The quantification of lipid droplets per cell was performed for measurement.
Oil Red staining was performed using an oil red O staining kit (Beyotime, China) with modifications. NP cells were fixed with 4% paraformaldehyde for 10 min and then stained with oil red O solution for 15 min at room temperature.

Chen X., Chen K., Hu J., Dong Y., Zheng M., Jiang J., Hu Q, & Zhang W. (2024). Palmitic acid induces lipid droplet accumulation and senescence in nucleus pulposus cells via ER-stress pathway. Communications Biology, 7, 539.

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