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BODIPY-Cholesterol

Catalog No.GC42964

BODIPY-Cholesterol Chemical Structure

BODIPY-Cholesterol is cholesterol tagged with a boron dipyrromethene difluoride (BODIPY) fluorophore used for monitoring sterol uptake and inter-organelle sterol flux in cells with excitation of 480 nm and emission of 508 nm.

Size Price Stock Qty
500μg
$69.00
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1mg
$131.00
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5mg
$259.00
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10mg
$485.00
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Sample solution is provided at 25 µL, 10mM.

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Protocol

Cell experiment [1]:

Cell lines

Human primary fibroblasts

Preparation Method

NPC1‐deficient GM3123 fibroblasts were incubated with Rhodamine‐dextran overnight to label terminal endocytic compartments, followed by incubation with 5 μg/mL (8.7 μm) BODIPY-Cholesterol in normal growth medium containing 5% fetal calf serum for 4 h, or 0.5 μm BODIPY-Cholesterol in medium containing 5% lipoprotein‐starved serum for 23 h.

Reaction Conditions

5 μg/mL, 4h

Applications

Time‐dependent partitioning of BODIPY-Cholesterol in the lysosomes of human primary fibroblasts.

Cell experiment [2]:

Cell lines

NIH 3T3

Preparation Method

NIH 3T3 were cultured in 24-well plates, incubated for 2 h with 2.5 µM BODIPY-cholesterol, and rinsed. Physiological buffer with or without 30 nM ShhN was added to the wells, and the BODIPY fluorescence intensity of the supernatants was measured after 1 h.

Reaction Conditions

2.5 µM; 2 h

Applications

The BODIPY fluorescence measured in the supernatants after 1 h was significantly lower in wells where ShhN protein was added compared to wells that did not contain ShhN.

References:

[1]. Hölttä-Vuori M, et al. Use of BODIPY-Cholesterol (TF-Chol) for Visualizing Lysosomal Cholesterol Accumulation. Traffic. 2016 Sep;17(9):1054-7.

[2]. Bidet M, et al. The hedgehog receptor patched is involved in cholesterol transport. PLoS One. 2011;6(9):e23834.

Background

BODIPY-Cholesterol is cholesterol tagged with a boron dipyrromethene difluoride (BODIPY) fluorophore used for monitoring sterol uptake and inter-organelle sterol flux in cells with excitation of 480 nm and emission of 508 nm.[1] The excretion of BODIPY cholesterol from late endoplasmic organelles depends on acidic lipase and Niemann pickc1 protein.[6]

In vitro, treatment with Bodipy-cholesterol in cells, prominent PM labeling is observed at 2–5 min; however, upon ≥30 min incubations, it is also observed the fluorescence labeling of intracellular structures.[2] In vitro efficacy test it suggested that accumulation of BODIPY-cholesterol in the media gave more reproducible values than assaying for the loss of the compound from the cells.[3] With 1 μg BODIPY-cholesterol analogs show a similar cellular localization in HeLa cells and exhibit similar cholesterol efflux properties from THP1 cells to HDL acceptors.[4] BODIPY-cholesterol efflux clearly increased when treatment of fibroblasts with the Hh pathway agonist SAG, which enhances Ptc protein expression, or over-expression of human Ptc in yeast.[5] Treatment with miR-758 inhibitor obviously increased ABCA1-dependent cholesterol efflux by BODIPY-Cholesterol efflux assay.[7] Micrographs of EPCs incubated with HDL labeld bodipy-cholesterol (50 µg/ml) 30 min, small cytoplasmic vesicles as well as large positive MVBs containing intraluminal microvesicles, tightly-packed with reaction products could be displayed. The internalized-HDL-derived bodipy-cholesterol was also spread within many of the stacked Golgi cisterns and the TGN.[8]

References:
[1].Wüstner D, et al. Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells. Chem Phys Lipids. 2016 Jan;194:12-28.
[2].Hölttä-Vuori M, et al. BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms. Traffic. 2008 Nov;9(11):1839-49.
[3].Sankaranarayanan S, et al. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol. J Lipid Res. 2011 Dec;52(12):2332-2340.
[4].Liu Z, et al. Synthesis of cholesterol analogues bearing BODIPY fluorophores by Suzuki or Liebeskind-Srogl cross-coupling and evaluation of their potential for visualization of cholesterol pools. Chembiochem. 2014 Sep 22;15(14):2087-96.
[5].Bidet M, et al. The hedgehog receptor patched is involved in cholesterol transport. PLoS One. 2011;6(9):e23834.
[6].Kanerva K, et al. LDL cholesterol recycles to the plasma membrane via a Rab8a-Myosin5b-actin-dependent membrane transport route. Dev Cell. 2013 Nov 11;27(3):249-62.
[7].Yao Y, et al. Glucagon-like peptide-1 contributes to increases ABCA1 expression by downregulating miR-758 to regulate cholesterol homeostasis. Biochem Biophys Res Commun. 2018 Mar 4;497(2):652-658.
[8].Srisen K, et al. Human endothelial progenitor cells internalize high-density lipoprotein. PLoS One. 2013 Dec 30;8(12):e83189.

Chemical Properties

Cas No. 878557-19-8 SDF
Synonyms BCh2
Chemical Name (T-4)-[(3β)-24-(3,5-dimethyl-1H-pyrrol-2-yl-κN)-24-(3,5-dimethyl-2H-pyrrol-2-ylidene-κN)chol-5-en-3-olato]difluoro-boron
Canonical SMILES O[C@H](C1)CC[C@@]2(C)C1=CC[C@]3([H])[C@]2([H])CC[C@@]4(C)[C@@]3([H])CC[C@]4([H])[C@H](C)CCC(C5=C(C)C=C(C)N5[B-]6(F)F)=C7[N+]6=C(C)C=C7C
Formula C36H51BF2N2O M.Wt 576.6
Solubility 0.5mg/ml in ethanol; 1mg/ml in DMSO, or in DMF Storage Store at -20°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Potential of BODIPY -Cholesterol for analysis of Cholesterol transport and diffusion in living cells

Chem Phys Lipids2016 Jan;194:12-28.PMID: 26291493DOI: 10.1016/j.chemphyslip.2015.08.007

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of Cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable Cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to Cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged Cholesterol analogs and in particular BODIPY -Cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY -tagged Cholesterol probes for analyzing intracellular Cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.

BODIPY -Cholesterol can be reliably used to monitor Cholesterol efflux from capacitating mammalian spermatozoa

Sci Rep2019 Jul 8;9(1):9804.PMID: 31285440DOI: 10.1038/s41598-019-45831-7

Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of Cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY -Cholesterol assay to quantify Cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY -Cholesterol assay was validated with endogenous Cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY -Cholesterol assay was able to detect and quantify Cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of Cholesterol efflux. Finally, the BODIPY -Cholesterol assay proved to be the superior method to quantify Cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing Cholesterol redistribution. Taken together, the BODIPY -Cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of Cholesterol efflux from spermatozoa during in vitro capacitation.

Liraglutide improves lipid metabolism by enhancing Cholesterol efflux associated with ABCA1 and ERK1/2 pathway

Cardiovasc Diabetol2019 Nov 9;18(1):146.PMID: 31706303DOI: 10.1186/s12933-019-0954-6

Background: Reverse Cholesterol transport (RCT) is an important cardioprotective mechanism and the decrease in Cholesterol efflux can result in the dyslipidemia. Although liraglutide, a glucagon like peptide-1 analogue, has mainly impacted blood glucose, recent data has also suggested a beneficial effect on blood lipid. However, the exact mechanism by which liraglutide modulates lipid metabolism, especially its effect on RCT, remain undetermined. Hence, the aim of the present study was to investigate the potential impacts and potential underlying mechanisms of liraglutide on the Cholesterol efflux in both db/db mice and HepG2 cells.
Methods: Six-week old db/db mice with high fat diet (HFD) and wild type mice were administered either liraglutide (200 μg/kg) or equivoluminal saline subcutaneously, twice daily for 8 weeks and body weight was measured every week. After the 8-week treatment, the blood was collected for lipid evaluation and liver was obtained from the mice for hematoxylin-eosin (HE) staining, red O staining and Western blotting. Cholesterol efflux was assessed by measuring the radioactivity in the plasma and feces after intraperitoneal injection of 3H-labeled Cholesterol . HepG2 Cells were treated with different concentrations of glucose (0, 5, 25, and 50 mmol/L) with or without liraglutide (1000 nmol/L) for 24 h. The intracellular Cholesterol efflux was detected by BODIPY -Cholesterol fluorescence labeling. Real-time PCR or Western blotting was used to examine the expression levels of ABCA1, ABCG1 and SR-B1.
Results: Liraglutide significantly decreased blood glucose, serum total Cholesterol (TC), triglyceride (TG) and low-density lipoprotein Cholesterol (LDL-C). It also reduced liver lipid deposition in db/db mice fed with HFD. Moreover, the movement of 3H-Cholesterol from macrophages to plasma and feces was significantly enhanced in db/db mice fed with HFD after liraglutide adminstration. In vitro study, liraglutide could promote the Cholesterol efflux of HepG2 cells under high glucose, and also increase the expression of ABCA1 by activating the ERK1/2 pathway.
Conclusions: Liraglutide could improve lipid metabolism and hepatic lipid accumulation in db/db mice fed with HFD by promoting reversal of Cholesterol transport, which was associated with the up-regulation of ABCA1 mediated by the ERK1/2 phosphorylation.

Use of BODIPY -labeled sphingolipid and Cholesterol analogs to examine membrane microdomains in cells

Histochem Cell Biol2008 Nov;130(5):819-32.PMID: 18820942DOI: 10.1007/s00418-008-0509-5

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in Cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (BODIPY ) labeled analogs of sphingolipids and Cholesterol to study such domains. We discuss the similarities between the behavior of BODIPY -Cholesterol and natural Cholesterol in artificial bilayers and in cultured cells, and the use of BODIPY -sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the BODIPY -fluorophore. The use of BODIPY -D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that BODIPY -sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.

Use of BODIPY -Cholesterol (TF-Chol) for Visualizing Lysosomal Cholesterol Accumulation

Traffic2016 Sep;17(9):1054-7.PMID: 27187581DOI: 10.1111/tra.12414

Dipyrromethene difluoride-Cholesterol (TopFluor-Cholesterol , TF-Chol) is a widely used Cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural Cholesterol in terms of membrane partitioning. However, the suitability of TF-Chol for detecting lysosomal Cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time-point both affect the membrane distribution and labeling pattern of TF-Chol, similarly as with radiolabeled Cholesterol . Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low-density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes.

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