BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry
显微镜观察BODIPY 493/503 染色中性脂滴以及用流式细胞仪量化
发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1912 浏览次数: 61612
评审: Lee-Hwa TaiYong TengVanesa Olivares-Illana
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参见作者原研究论文
The authors used this protocol in:
Jun 2015
Abstract
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.
Keywords: Neutral lipid (中性脂质)
Lipid droplet (脂滴)
BODIPY (BODIPY)
Materials and Reagents
- 35 mm cell culture dish/well
- Flow cytometry tube with cell strainer cap (Corning, catalog number: 352235 )
- 15 ml conical tube
- 35 μm filter
- FACS tube
- Glass slides
- Circular cover slip (12 mm #1 circular) (VWR International, catalog number: 101415-528 )
- Cell line of interest
Note: For this protocol, we utilize the A498 clear cell renal cell carcinoma cell line (ATCC, catalog number: A-498 ), but this method can be readily performed with other cell lines. - Oleic acid (OA) (100 mg/ml with 10% BSA in DPBS) (Sigma-Aldrich, catalog number: O3008 )
- Bovine serum albumin (BSA) (10% in DPBS, low endotoxin, fatty acid free, suitable for cell culture, sterile-filtered) (Sigma-Aldrich, catalog number: A1595-50ML )
- 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D3922 )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
- Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco®, catalog number: 10010031 )
- Trypsin-EDTA (0.25%) with phenol red (Thermo Fisher Scientific, Gibco®, catalog number: 25200-056 )
- HEPES
- NaCl
- CaCl2
- Dulbecco’s modified Eagle’s medium with high glucose and L-glutamine (Thermo Fisher Scientific, Fisher Scientific, catalog number: SH3024301 ) or other medium appropriate for cell line of interest
- Fetal bovine serum (FBS) (Gemini Bio-Products, FoundationTM, catalog number: 900-108 ) or other FBS suitable for cell line of interest
Note: For this protocol, cells were grown in DMEM + 10% FBS. - 4% paraformaldehyde (PFA)
- Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36935 )
- Collagen I, rat tail (100 mg) (Corning, catalog number: 354236 )
- 2 μM BODIPY staining solution (see Recipes)
- 10x flow cytometry buffer (see Recipes)
Note: 10x buffer can also be purchased [10x Annexin binding buffer (BD, catalog number: 556454 )].
Equipment
- Forceps
- Flow cytometer equipped with 488 mm laser and filter sets for measuring FITC or GFP (i.e., 533/30)
Note: In this study, a BD Accuri C6 instrument was used.
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
细胞生物学 > 细胞染色 > 脂质
生物化学 > 脂质 > 脂质测定
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