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BODIPY 493/503 Sale

(Synonyms: 4,4-二氟-1,3,5,7,8-五甲基-4-硼-3A,4A-二氮杂-S-茚烯,Pyrromethene 546; BDP 493/503 lipid stain) 目录号 : GC42959

BODIPY 493/503是一种亲脂性荧光染料,发出明亮的绿色荧光,被广泛用于标记脂质滴(激发/发射:493/503纳米)。

BODIPY 493/503 Chemical Structure

Cas No.:121207-31-6

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实验参考方法

Protocol for BODIPY 493/503 staining with flow cytometry [1]
1. Grow cells under culture conditions relevant for the study. (For example, 50,000 A498 cells in 35 mm well.)
Note: Overnight incubation of cells with 30 μM oleic acid can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control.
2. At the time-point of interest, prepare 2 μM BODIPY 493/503 staining solution in PBS.
3. Wash cells with a quick rinse using 3 ml PBS to remove media/serum.
4. Incubate on BODIPY 493/503 staining solution in the dark for 15 min at 37 °C. Include an unstained control for flow cytometry.
5. Wash cells with a quick rinse using 3 ml PBS to remove staining solution.
6. Trypsinize cells to generate a single cell suspension. For the A498 cell line used in this protocol, cells were incubated with Trypsin-EDTA (0.25%) for 5 min at 37 °C.
7. Add 5 ml of PBS and transfer cell suspension to a 15 ml conical tube.
8. Pellet cells at 250×g, 5 min, 4 °C.
9. Aspirate supernatant, wash the cell pellet with a quick rinse using 3 ml PBS, and pellet cells at 250 ×g, 5 min, 4 °C.
10. Carefully aspirate the supernatant and resuspend cells in 300 μl 1x flow cytometry buffer.
11. Pass cell suspension through a 35 μm filter into a FACS tube.
12. Perform flow cytometry. Obtain a minimum of 10,000 events per condition.
13. The investigator can analyze data as mean fluorescence or display the data as a histogram.

Protocol for BODIPY 493/503 staining with microscopy [1]
1. Autoclave coverslips in a glass bottle.
2. In the tissue culture hood, place coverslips into 35 mm cell culture dishes.
3. Prepare 2 mg/ml collagen solution in PBS.
4. Treat the coverslips with collagen to promote cell adherence. Add 3 ml collagen solution to culture dishes and incubate at 37 °C for 30 min.
Note: Use forceps to ensure that coverslips are flush with the bottom of the culture dish, eliminating any air bubbles that may be under the cover slips.
5. Aspirate the collagen solution.
6. Wash with PBS.
7. Add PBS to culture dishes and place under UV light in the culture hood to sterilize.
8. Plate cells into culture dishes containing the coverslips. The optimal cell number should be determined to achieve confluence of 30-50% at the time of staining to permit proper imaging. For A498 cells used in this protocol, 100,000 cells were plated in 35 mm wells to permit staining at 48 h post plating.
9. Incubate under the culture conditions relevant to your experiment.
a. For this protocol, A498 cells were incubated in DMEM (high glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS at 37 °C.
b. Overnight incubation of cells with 30 μM oleic acid with BSA can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control.
10. At the time-point of interest, prepare 2 μM BODIPY 493/503 staining solution in PBS.
a. For this protocol, A498 cells were stained 48 h after plating, after an overnight incubation with BSA or BSA + oleic acid.
11. Wash cells with 3 ml PBS.
12. Incubate on 3 ml staining solution for 15 min at 37 °C.
Note: From this point, protect samples from light as much as possible.
13. Wash twice in 3 ml PBS.
14. Fix cells in 3 ml 4% PFA for 30 min at room temperature.
15. Remove 4% PFA.
16. Wash samples 3 x 5 min in PBS.
17. Use forceps to mount cover slips onto glass slides.
Use forceps to pick up cover slips and place onto the drop of mounting solution, ensuring that the side that side with cells is placed face down onto the glass slides.
18. Allow the mounting solution to cure overnight at room temperature.
19. Immediately image cells.
BODIPY 493/503 photobleaches rapidly. Including 200 ng/ml of BODIPY 493/503 in the medium during imaging can minimize this problem. Additionally, using the stable Hoechst staining in the blue channel to find, focus, and center fields before imaging BODIPY 493/503 in the green channel diminishes photobleaching [2] .
2 μM BODIPY staining solution:
a. Prepare 5 mM BODIPY stock solution
Dissolve 1.3 mg BODIPY in 1 ml DMSO and can be stored at -20 °C, protect from light.
b. 2 μM BODIPY staining solution can be prepared by diluting stock solution 1:2,500 in PBS.

This protocol only provides a guideline, and should be modified according to your specific needs

References:
[1]. Qiu, B. and Simon, M. C. (2016). BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry. Bio-protocol 6(17): e1912. DOI: 10.21769/BioProtoc.1912.
[2]. Listenberger, L.L., Studer, A.M., Brown, D.A., and Wolins, N.E. 2016. Fluorescent detection of lipid droplets and associated proteins. Curr. Protoc. Cell Biol. 71:4.31.1-4.31.14. doi: 10.1002/cpcb.7

产品描述

BODIPY 493/503, a lipophilic fluorescence dye, emits bright green fluorescence has been used extensively for lipid droplet labeling (Ex/Em: 493/503 nm) [1,2]. BODIPY 493/503 is compatible with epifluorescent, confocal, and two-photon microscopy, as well as flow cytometry, and can be used for live and fixed cell applications.

BODIPY 493/503是一种亲脂性荧光染料,发出明亮的绿色荧光,被广泛用于标记脂质滴(激发/发射:493/503纳米)[1,2]。BODIPY 493/503适用于表面荧光显微镜、共聚焦显微镜、双光子显微镜以及流式细胞术,并可用于活体和固定细胞应用。

References:
[1]. Ohsaki Y, Shinohara Y, Suzuki M, et al. A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy[J]. Histochemistry and cell biology, 2010, 133(4): 477-480.
[2]. Listenberger, L.L., Studer, A.M., Brown, D.A., and Wolins, N.E. 2016. Fluorescent detection of lipid droplets and associated proteins. Curr. Protoc. Cell Biol. 71:4.31.1-4.31.14. doi: 10.1002/cpcb.7

Chemical Properties

Cas No. 121207-31-6 SDF
别名 4,4-二氟-1,3,5,7,8-五甲基-4-硼-3A,4A-二氮杂-S-茚烯,Pyrromethene 546; BDP 493/503 lipid stain
化学名 (T-4)-[2-[1-(3,5-dimethyl-2H-pyrrol-2-ylidene-κN)ethyl]-3,5-dimethyl-1H-pyrrolato-κN]difluoro-boron
Canonical SMILES [F-][B+3]([N-]1C2=C(C)C=C1C)([N](C(C(C)=C3)=C2C)=C3C)[F-]
分子式 C14H17BF2N2 分子量 262.1
溶解度 Soluble in DMSO, Soluble in DMF, Soluble in Chloroform, Soluble in Methanol 储存条件 Store at -20°C, protect from light
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储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。
为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。
Shipping Condition 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。

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1 mg 5 mg 10 mg
1 mM 3.8153 mL 19.0767 mL 38.1534 mL
5 mM 0.7631 mL 3.8153 mL 7.6307 mL
10 mM 0.3815 mL 1.9077 mL 3.8153 mL
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Research Update

BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry

Bio Protoc 2016 Sep 5;6(17):e1912.PMID:28573161DOI:10.21769/BioProtoc.1912.

Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis (Greenberg et al., 2011). For example, neutral lipid storage is enhanced upon increased synthesis or uptake of lipid species. On the other hand, extracellular signals can enhance the release of lipid species packaged within neutral LDs. Thus, the investigation of topics involving lipid metabolism may require the assessment of cellular neutral lipid content. In this protocol, we describe the use of the fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) to facilitate quantification of neutral lipid content by flow cytometry and observation of LDs by microscopy.

SIRT1 prevents cigarette smoking-induced lung fibroblasts activation by regulating mitochondrial oxidative stress and lipid metabolism

J Transl Med 2022 May 14;20(1):222.PMID:35568871DOI:10.1186/s12967-022-03408-5.

Background: Cigarette smoking (CS) is a strong risk factor for idiopathic pulmonary fibrosis (IPF). It can activate lung fibroblasts (LF) by inducing redox imbalance. We previously showed that clearing mitochondrial reactive oxygen species (mtROS) protects against CS-induced pulmonary fibrosis. However, the precise mechanisms of mtROS in LF need further investigation. Here we focused on mtROS to elucidate how it was regulated by CS in LF and how it contributed to LF activation. Methods: We treated cells with 1% cigarette smoking extract (CSE) and examined mtROS level by MitoSOX™ indicator. And the effect of CSE on expression of SIRT1, SOD2, mitochondrial NOX4 (mtNOX4), fatty acid oxidation (FAO)-related protein PPARα and CPT1a and LF activation marker Collagen I and α-SMA were detected. Nile Red staining was performed to show cellular lipid content. Then, lipid droplets, autophagosome and lysosome were marked by BODIPY 493/503, LC3 and LAMP1, respectively. And lipophagy was evaluated by the colocalization of lipid droplets with LC3 and LAMP1. The role of autophagy on lipid metabolism and LF activation were explored. Additionally, the effect of mitochondria-targeted ROS scavenger mitoquinone and SIRT1 activator SRT1720 on mitochondrial oxidative stress, autophagy flux, lipid metabolism and LF activation were investigated in vitro and in vivo. Results: We found that CS promoted mtROS production by increasing mtNOX4 and decreasing SOD2. Next, we proved mtROS inhibited the expression of PPARα and CPT1a. It also reduced lipophagy and upregulated cellular lipid content, suggesting lipid metabolism was disturbed by CS. In addition, we showed both insufficient FAO and lipophagy resulted from blocked autophagy flux caused by mtROS. Moreover, we uncovered decreased SIRT1 was responsible for mitochondrial redox imbalance. Furthermore, we proved that both SRT1720 and mitoquinone counteracted the effect of CS on NOX4, SOD2, PPARα and CPT1a in vivo. Conclusions: We demonstrated that CS decreased SIRT1 to activate LF through dysregulating lipid metabolism, which was due to increased mtROS and impaired autophagy flux. These events may serve as therapeutic targets for IPF patients.

Fluorescent detection of lipid droplets and associated proteins

Curr Protoc Cell Biol 2007 Jun;Chapter 24:Unit 24.2.PMID:18228510DOI:10.1002/0471143030.cb2402s35.

Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.

SNX10-mediated degradation of LAMP2A by NSAIDs inhibits chaperone-mediated autophagy and induces hepatic lipid accumulation

Theranostics 2022 Feb 21;12(5):2351-2369.PMID:35265214DOI:10.7150/thno.70692.

Rationale: While some non-steroidal anti-inflammatory drugs (NSAIDs) are reported to induce hepatic steatosis, the molecular mechanisms are poorly understood. This study presented the mechanism by which NSAIDs induce hepatic lipid accumulation. Methods: Mouse primary hepatocytes and HepG2 cells were used to examine the underlying mechanism of NSAID-induced hepatic steatosis. Lipid accumulation was measured using Nile-red assay and BODIPY 493/503. The activity of chaperone-mediated autophagy (CMA) was determined by western blotting, qRT-PCR, and confocal imaging. The effect of NSAID on CMA inhibition was evaluated in vivo using diclofenac and CMA activator (AR7) administered mice. Results: All tested NSAIDs in this study accumulated neutral lipids in hepatocytes, diclofenac having demonstrated the most potency in that regard. Diclofenac-induced lipid accumulation was confirmed in both mouse primary hepatocytes and the liver of mice. NSAIDs inhibited CMA, as reflected by the decreased expression of lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A) protein, the increased expression of CMA substrate proteins such as PLIN2, and the decreased activity of photoactivatable KFERQ-PAmCherry reporter. Reactivation of CMA by treatment with AR7 or overexpression of LAMP2A inhibited diclofenac-induced lipid accumulation and hepatotoxicity. Upregulation of sorting nexin 10 (SNX10) via the CHOP-dependent endoplasmic reticulum stress response and thus maturation of cathepsin A (CTSA) was shown to be responsible for the lysosomal degradation of LAMP2A by diclofenac. Conclusion: We demonstrated that NSAIDs induced SNX10- and CTSA-dependent degradation of LAMP2A, thereby leading to the suppression of CMA. In turn, impaired CMA failed to degrade PLIN2 and disrupted cellular lipid homeostasis, thus leading to NSAID-induced steatosis and hepatotoxicity.

Regulatory roles of external cholesterol in human airway epithelial mitochondrial function through STARD3 signalling

Clin Transl Med 2022 Jun;12(6):e902.PMID:35678098DOI:10.1002/ctm2.902.

Background: Hypercholesterolemia is found in patients with chronic lung inflammation, during which airway epithelial cells play important roles in maintenance of inflammatory responses to pathogens. The present study aims at molecular mechanisms by which cholesterol changes airway epithelial sensitivity in response to smoking. Methods: Human bronchial epithelial cells (HBEs) were stimulated with cigarette smoke extract (CSE) and mice were exposed to CS/lipopolysaccharide (LPS) as models in vitro and in vivo. Severe COPD patients and healthy volunteers were also enrolled and the level of cholesterol in plasma was detected by metabolomics. Filipin III and elisa kits were used to stain free cholesterol. Mitochondrial function was detected by mitotracker green, mitotracker green, and Seahorse. Mitochondrial morphology was detected by high content screening and electron microscopy. The mRNA and protein levels of mitochondrial dynamics-related proteins were detected by RT-qPCR and Western blot,respectively. BODIPY 493/503 was used to stain lipid droplets. Lipidomics was used to detect intracellular lipid components. The mRNA level of interleukin (IL)-6 and IL-8 were detected by RT-qPCR. Results: We found that the cholesterol overload was associated with chronic obstructive pulmonary disease (COPD) and airway epithelia-driven inflammation, evidenced by hypercholesterolemia in patients with COPD and preclinical models, alteration of lipid metabolism-associated genes in CSE-induced airway epithelia and production of ILs. External cholesterol altered airway epithelial sensitivity of inflammation in response to CSE, through the regulation of STARD3-MFN2 pathway, cholesterol re-distribution, altered transport and accumulation of cholesterol, activities of lipid transport regulators and disorder of mitochondrial function and dynamics. MFN2 down-regulation increased airway epithelial sensitivity and production of ILs after smoking, at least partially by injuring fatty acid oxidation and activating mTOR phosphorylation. Conclusions: Our data provide new insights for understanding molecular mechanisms of cholesterol-altered airway epithelial inflammation and for developing diagnostic biomarkers and therapeutic targets to improve patient outcomes.