Neutral lipids and lipid droplets. BODIPY 493/503 methyl bromide is a lipophilic dye that partitions into neutral lipid compartments within cells, particularly lipid droplets. It does not have a specific protein target but rather accumulates in neutral lipid droplets based on its hydrophobicity. The “493/503” designation refers to its excitation/emission maxima. The methyl bromide group can be used to chemically conjugate the dye to other molecules, but in its native form, it is used as a direct stain for lipid droplets and neutral lipids. Because BODIPY 493/503 is less polar than other BODIPY dyes, it preferentially stains neutral lipids over polar lipids (such as membrane phospholipids). This makes it highly specific for lipid droplets, which are composed primarily of neutral lipids (triglycerides, cholesterol esters). The dye is used to visualize lipid droplet size, number, and distribution in cells, and to study lipid metabolism, obesity, fatty liver disease, and atherosclerosis. It is also used as a tracer for fatty acid uptake and metabolism, as a fluorescent fatty acid analog (the methyl bromide group may be replaced by a fatty acid chain, or the dye itself is used as a stand-in for a fatty acid due to its hydrophobic nature). In in vitro binding assays, the dye can be used to label isolated lipid droplets or synthetic lipid emulsions. It is also used in fluorescence-based assays to measure lipid droplet formation, lipolysis, and lipid exchange processes. The dye is cell-permeable and can be used for live-cell imaging as well as for fixed-cell staining.

Guidelines for usage 1. BODIPY493/503 methyl bromide working solution composition 1.1 Prepare the stock solution by adding 1 milligram of BODIPY493/503 methyl bromide to 10 mM of anhydrous DMSO (382 μL). Note: To generate a 1-10 μM BODIPY493/503 methyl bromide working solution, it is advised to aliquot the BODIPY493/503 methyl bromide storage solution into the 1.2 working solution rather than using pre-prepared serum-free cell culture medium or PBS. Please prepare the BODIPY493/503 methyl bromide working solution for usage by adjusting its concentration to the current circumstances. 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. 2.2 Add 1 mL of BODIPY493/503 methyl bromide working solution and stir for 5–30 minutes when the cell density is 1×106/mL. 2.3 Centrifuge at 400g for 3–4 minutes and discard the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Adherent cell staining: 3.1 Grow the adherent cells on sterile coverslips. 3.2 Cover the cells fully with 100 μL of the dye working solution from step 3.3, give them a gentle shake, and sterilize for a duration of 5 to 30 minutes. 3.4 Aspirate the dye working solution, rinse with culture medium two or three times for five minutes each, and use a flow cytometer or fluorescence microscope to monitor. Conditions for storage: Keep for a year at -20°C and out of direct sunlight. Observations 1. Kindly modify the BODIPY493/503 methyl bromide working solution concentration and the left-hand time interval based on the current circumstances. 2. It is advised that the experiment include an acute control. Before doing any further tests, the cells need to be treated to a 30 μM oleic acid carrier for 8 hours. 3. This product is not intended for use in clinical diagnosis or treatment; rather, it should only be used by experts conducting scientific research. 4. When working, please wear immediate gloves and a lab coat for your health and safety.

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In vitro, BODIPY 493/503 methyl bromide is widely used for the specific staining of neutral lipids and lipid droplets in cells and tissues. At working concentrations of 1-10 uM in serum-free medium or PBS, cells become brightly stained within 5-30 minutes, with a punctate pattern corresponding to lipid droplets. The dye is highly specific for neutral lipids and does not significantly stain other cellular membranes, providing high contrast. It is compatible with both live-cell and fixed-cell imaging (after mild formaldehyde fixation). The dye can be used to quantify cellular lipid content by fluorescence microscopy (e.g., integrated fluorescence intensity per cell) or by flow cytometry. It is also used to study lipid droplet dynamics, such as their formation, fusion, fission, and degradation during lipophagy. The dye can be combined with other fluorescent markers (e.g., for mitochondria, ER, or lysosomes) for multi-color imaging. In biochemical assays, BODIPY 493/503 methyl bromide can be used to label purified lipid droplets or lipoprotein particles for in vitro binding or uptake studies. For example, the dye can be incorporated into artificial lipid droplets (by mixing with triglycerides and phospholipids) and then used to study the interaction of lipid droplets with cells or proteins. The dye is also used as a fluorescent probe in high-throughput screening assays for modulators of lipid droplet formation. It is an excellent tool for studying lipid metabolism disorders. The bromomethyl group could potentially be used to conjugate the dye to other molecules, but for most staining applications, the unmodified dye is used directly. In vitro, the dye is typically used at 1-10 uM, with incubation times of 5-30 minutes. For fixed samples, cells are often fixed with 4% paraformaldehyde before staining, or after staining. The dye is compatible with immunofluorescence, but the methanol permeabilization step should be avoided if possible as it may extract lipids and remove the dye. For live-cell imaging, the dye should be used in serum-free medium, as serum contains albumin and lipoproteins that may bind and remove the dye from the cells. An optimal staining concentration may need to be determined empirically for each cell type and condition. BODIPY 493/503 methyl bromide is more specific for neutral lipids compared to other BODIPY dyes such as BODIPY 493/503 (without the methyl bromide group?) or Nile Red. Its spectral properties (Ex/Em=493/503 nm) are well-suited for GFP/FITC filter sets.

In vivo, BODIPY 493/503 methyl bromide can be used to image lipid droplets and lipid distribution in small transparent animal models, such as zebrafish larvae. The dye is added to the aquarium water (1-10 uM) or microinjected into zebrafish embryos or larvae. After uptake, the dye accumulates in lipid droplets, enabling live imaging of lipid metabolism, yolk absorption, and the effects of drugs or genetic manipulations on lipid homeostasis. In mouse models, the dye can be administered systemically (intravenous or intraperitoneal injection) to label lipid droplets in various tissues, but the green fluorescence limits tissue penetration. Topical application or injection into specific tissues (e.g., subcutaneous fat, tumors) can be used for ex vivo imaging of tissue sections. The dye can also be used for ex vivo imaging of lipid-rich tissues such as the liver, adipose tissue, and atherosclerotic plaques. After systemic administration, the dye may be quickly cleared from the bloodstream and taken up by hepatocytes and adipocytes. Detailed in vivo activity data is limited, as the dye is primarily used in vitro or ex vivo. No clinical applications are approved.

For a cell-free lipid binding assay, BODIPY 493/503 methyl bromide can be used to assess its interaction with isolated lipid droplets or artificial lipid emulsions. Lipid droplets are isolated from cells (e.g., adipocytes or hepatocytes) by ultracentrifugation. Alternatively, synthetic lipid droplets are prepared by emulsifying a lipid mixture (e.g., triolein or triglycerides) with a surfactant (e.g., phosphatidylcholine) in buffer. The dye is dissolved in DMSO to a stock concentration of 1-10 mM. The isolated lipid droplets or synthetic lipid emulsions are diluted in PBS (lipid concentration 0.1-1 mg/mL). The dye is added to the lipid emulsion at various concentrations (0.1-10 uM). The mixture is incubated for 10-30 minutes at room temperature. Fluorescence intensity is measured using a fluorimeter or plate reader (excitation 488 nm, emission 510-530 nm). An increase in fluorescence intensity (relative to dye in buffer without lipids) indicates binding to lipids. The binding constant (Kd) can be estimated by titrating increasing lipid concentrations and fitting the fluorescence intensity change. Alternatively, the partition coefficient (logP) can be determined by measuring the dye's distribution between an aqueous buffer and an organic solvent (e.g., octanol) or lipid phase. This assay provides information about the lipophilic nature of the probe and its specificity for neutral lipids versus other lipid classes.

For cellular staining of lipid droplets, the following protocol is commonly used. Prepare a 1-10 mM stock solution of BODIPY 493/503 methyl bromide in DMSO. For working solution, dilute the stock solution in pre-warmed serum-free cell culture medium or PBS to a final concentration of 1-10 uM (adjust as needed for specific applications). For adherent cells: culture cells on coverslips or in 96-well plates to 70-80% confluence. Remove the culture medium and wash cells once with PBS. Add the dye working solution (100-200 uL per well for a 24-well plate, or enough to cover the cells) and incubate for 5-30 minutes at 37degC or room temperature, protected from light. After incubation, remove the dye solution and wash cells 2-3 times with PBS (5 minutes each wash). For live-cell imaging, add fresh phenol red-free culture medium or PBS and image immediately using a fluorescence microscope with excitation at 488 nm and emission at 505-530 nm (FITC/GFP filter set). For fixed-cell imaging, after staining and washing, cells can be fixed with 4% paraformaldehyde for 10-15 minutes at room temperature, washed, and mounted with an anti-fade mounting medium (optionally containing DAPI to counterstain nuclei). The dye is compatible with formaldehyde fixation but avoid methanol fixation as it may extract lipids. For suspension cells: collect cells by centrifugation (300-400 × g, 3-5 min), wash once with PBS, resuspend in dye working solution (1×10⁶ cells/mL) and incubate for 5-30 minutes at room temperature, protected from light. Wash 2-3 times with PBS, resuspend in PBS or serum-free medium, and analyze by flow cytometry (488 nm laser, 530/30 nm emission filter) or prepare cytospin slides for microscopy. For tissue sections: frozen sections (10-20 um) are washed with PBS, incubated with dye working solution (1-10 uM in PBS) for 15-30 minutes at room temperature, washed with PBS, and mounted with an anti-fade medium. For paraffin-embedded sections, deparaffinize, rehydrate, and then stain (though lipid extraction during processing may reduce signal; frozen sections are preferred for lipid staining). The dye should be stored at -20degC in the dark as a powder, and stock solutions in DMSO should be stored at -20degC for up to 6 months. Avoid repeated freeze-thaw cycles. The staining concentration and time may need to be optimized for different cell types. Positive controls: cells treated with oleic acid (100-300 uM) to induce lipid droplet accumulation. Negative controls: cells treated with lipase inhibitors or cultured under serum-free conditions to minimize lipid droplets. This product is intended for research use only and not for clinical or diagnostic applications.

For in vivo lipid droplet imaging in zebrafish, a stock solution of BODIPY 493/503 methyl bromide (10 mM in DMSO) is diluted in embryo water (or E3 medium) to a final concentration of 1-10 uM (final DMSO concentration ≤0.1%). Zebrafish larvae (e.g., 3-7 days post-fertilization, dpf) are transferred to a 6-well plate or small dish containing the dye solution. Larvae are incubated in the dark for 30-60 minutes at 28degC. After staining, larvae are washed 3 times with fresh E3 medium (5 minutes each) and then anesthetized with 0.016% tricaine (MS-222) for imaging. Larvae are placed on a glass slide with a drop of methylcellulose to immobilize them, and images are acquired using a fluorescence stereomicroscope or confocal microscope with a GFP/FITC filter set (excitation 488 nm, emission 515-530 nm). For mouse studies, BODIPY 493/503 methyl bromide can be dissolved in sterile PBS containing <10% DMSO and 1% Tween-80 at a concentration of 0.5-2 mg/mL. For intraperitoneal injection, mice receive 100-200 uL of the solution (dose 2-10 mg/kg). For intravenous injection (tail vein), the same dose can be administered. Mice are allowed to circulate the dye for 1-6 hours, then euthanized. Tissues of interest (e.g., liver, adipose tissue, kidney) are harvested, fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and frozen in OCT compound for cryosectioning. Tissue sections (10-20 um) are stained with DAPI and imaged by fluorescence microscopy. For whole-body imaging (using a mouse imaging system), green excitation/emission may be used, but due to high background and poor tissue penetration, this is not optimal; red/near-infrared dyes are recommended for whole-body imaging in mice. The compound is not currently used for clinical imaging in humans. The dye is for research use only, and all applicable animal care and use guidelines must be followed.

Pharmacokinetic data for BODIPY 493/503 methyl bromide (MW 341.00, logP ~5) are limited due to its primary use as a research stain. Based on its high lipophilicity, the dye is likely to be rapidly cleared from the circulation (half-life of minutes) after intravenous administration and will be taken up by the liver, adipose tissue, and other lipid-rich tissues. It may be stored in lipid droplets and slowly eliminated over days to weeks. The dye is not significantly metabolized, but the methyl bromide group may be susceptible to nucleophilic substitution, leading to conjugation with endogenous thiols. However, for staining purposes, the dye is used as a direct stain, and PK data are not typically required. Researchers should be aware that the dye is not intended for systemic use in humans and may accumulate in tissues if injected. For accurate quantitative biodistribution studies, the dye can be extracted from tissues with organic solvent (e.g., methanol/chloroform) and analyzed by HPLC with fluorescence detection. Due to the lack of published PK data, it is recommended that users conduct their own studies if needed. The dye's fluorescence is stable in the dark, but photobleaching can occur under prolonged illumination. For live-cell imaging, minimize light exposure to avoid phototoxicity and bleaching.

Toxicological data for BODIPY 493/503 methyl bromide are limited. In vitro, the dye is generally non-toxic at typical working concentrations (1-10 uM) over short exposure times (5-60 minutes), as assessed by cell viability assays (MTT, LDH). However, at higher concentrations (>50 uM) or prolonged exposure (>24 hours), some cytotoxicity may be observed, possibly due to the effects of the bromomethyl group (which is an alkylating agent) on cellular proteins and DNA. The dye should be handled with caution. The methyl bromide group is potentially genotoxic, as it can alkylate DNA. Therefore, appropriate safety measures should be taken: use gloves, lab coat, and eye protection; avoid inhalation of dust or aerosol; work in a well-ventilated area or fume hood when handling the powder or preparing stock solutions; avoid skin contact. The dye should be stored at -20degC, protected from light and moisture. The compound is for research use only and is not intended for human diagnostic or therapeutic use. Ensure personal safety with lab attire and disposable gloves while handling. Dispose of waste according to local regulations. No chronic toxicity or carcinogenicity data are available.

[1]. Synthesis and spectral properties of cholesterol- and FTY720-containing boron dipyrromethene dyes. Synthesis and spectral properties of cholesterol- and FTY720-containing boron dipyrromethene dyes. J Org Chem. 2007 Oct 26;72(22):8376-82.

[2]. BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry. Bio Protoc. 2016 Sep 5;6(17):e1912.

BODIPY 493/503 methyl bromide is a research-grade fluorescent dye with no approved clinical applications. It is supplied as a solid powder (color: orange to red) with a purity of ≥97% (typically 97-98%). The molecular weight is 341.00, and the formula is C14H16BBrF2N2. The dye is soluble in DMSO (10-20 mg/mL) and other organic solvents (DMF, ethanol, methanol), and should be stored as a powder at -20degC for up to 3 years, protected from light. Stock solutions (1-10 mM) in DMSO should be stored at -20degC in the dark for up to 6 months; avoid repeated freeze-thaw cycles. This dye is a highly specific and bright lipid droplet stain, widely used in cell biology to study lipid metabolism, obesity, fatty liver disease, diabetes, and cancer biology. Its excitation/emission maxima are 493/503 nm, providing a bright green fluorescence that can be distinguished from red or far-red fluorophores. The methyl bromide group is a potential handle for chemical modification, but for most staining applications, the unmodified dye is used directly. The dye is also known as BODIPY 493/503 methyl bromide and may be listed under synonyms such as 8-Bromomethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene. It is intended solely for scientific research by professionals and is not for clinical, food, or drug use. For optimal results, the dye should be protected from light during handling and storage. Always consult the product data sheet and primary literature for specific experimental details.

C14H16BN2F2BR

341.00204

340.055

216434-81-0

71774308

Brown to reddish brown solid powder

3.201

0

3

1

20

525

0

[B-]1(N2C(=CC(=C2C(=C3[N+]1=C(C=C3C)C)CBr)C)C)(F)F

JLSHPOSUMKVMAP-UHFFFAOYSA-N

InChI=1S/C14H16BBrF2N2/c1-8-5-10(3)19-13(8)12(7-16)14-9(2)6-11(4)20(14)15(19,17)18/h5-6H,7H2,1-4H3

8-(bromomethyl)-2,2-difluoro-4,6,10,12-tetramethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~10 mg/mL (~29.33 mM)

Solubility in Formulation 1: 1 mg/mL (2.93 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)