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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   

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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

  1. 35 mm cell culture dish/well
  2. Flow cytometry tube with cell strainer cap (Corning, catalog number: 352235 )
  3. 15 ml conical tube
  4. 35 μm filter
  5. FACS tube
  6. Glass slides
  7. Circular cover slip (12 mm #1 circular) (VWR International, catalog number: 101415-528 )
  8. 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.
  9. Oleic acid (OA) (100 mg/ml with 10% BSA in DPBS) (Sigma-Aldrich, catalog number: O3008 )
  10. Bovine serum albumin (BSA) (10% in DPBS, low endotoxin, fatty acid free, suitable for cell culture, sterile-filtered) (Sigma-Aldrich, catalog number: A1595-50ML )
  11. 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 )
  12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  13. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco®, catalog number: 10010031 )
  14. Trypsin-EDTA (0.25%) with phenol red (Thermo Fisher Scientific, Gibco®, catalog number: 25200-056 )
  15. HEPES
  16. NaCl
  17. CaCl2
  18. 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
  19. 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. 
  20. 4% paraformaldehyde (PFA)
  21. Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36935 )
  22. Collagen I, rat tail (100 mg) (Corning, catalog number: 354236 )
  23. 2 μM BODIPY staining solution (see Recipes)
  24. 10x flow cytometry buffer (see Recipes)
    Note: 10x buffer can also be purchased [10x Annexin binding buffer (BD, catalog number: 556454 )].

Equipment

  1. Forceps
  2. 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

  1. BODIPY staining for flow cytometry
    1. Grow cells under culture conditions relevant for the study. A 35 mm dish/well is sufficient for the cell numbers required in this assay. For our assays, 50,000 A498 cells in 35 mm well were sufficient.
      1. 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 staining solution in PBS. The volume of staining solution required for each sample corresponds to the volume of media used for culturing cells. 
    3. Wash cells with a quick rinse using 3 ml PBS to remove media/serum.
    4. Incubate on BODIPY staining solution in the dark for 15 min at 37 °C. Include an unstained control for flow cytometry.
      Note: From this point, protect samples from light as much as possible.
    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 x 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 x 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 (Figure 1A) or display the data as a histogram (Figure 1B). 

  2. BODIPY staining for microscopy
    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.
      1. For this protocol, A498 cells were incubated in DMEM (high glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS at 37 °C. 
      2. 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 staining solution in PBS. 
      1. 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.
      1. Add a drop of Prolong® Gold antifade reagent with DAPI onto slide.
      2. 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 mounting solution.
    18. Allow the mounting solution to cure overnight at room temperature.
    19. Slides can be stored at 4 °C or imaged immediately (Figure 1C).


      Figure 1. Quantification and visualization of intracellular neutral LDs. A. A498 cells were grown overnight in the presence of BSA (0.2%) or 30 μM oleic acid with BSA. The cells were analyzed according to the section titled “BODIPY staining for flow cytometry”. For each sample, mean FL-1 area was normalized to the mean FL-1 area for the BSA treated samples. Note that oleic acid increased FL-1 area. P-value was determined by an unpaired student’s t-test. ***P < 0.001. B. Representative histograms of cells described in (A). C. A498 cells grown on coverslips were treated overnight with BSA (0.2%) or 30 μM oleic acid with BSA. The cells were analyzed according to the section titled “BODIPY staining for microscopy”. Scale bar = 5 μm.

Recipes

  1. 2 μM BODIPY staining solution
    1. Prepare 5 mM BODIPY stock solution
      Dissolve 1.3 mg BODIPY in 1 ml DMSO and can be stored at -20 °C.
    2. 2 μM BODIPY staining solution can be prepared by diluting stock solution 1:2,500 in PBS.
  2. 10x flow cytometry buffer
    0.1 M HEPES (pH 7.4)
    1.4 M NaCl
    25 mM CaCl2
    Notes:
    1. 10x buffer can be stored at 4 °C.
    2. Dilute to 1x using MilliQ water prior to use.

Acknowledgments

This work was supported by NIH grant 2-P01-CA104838 to M.C.S, and NIH fellowship 5-F30-CA177106 to B.Q.

References

  1. Greenberg, A. S., Coleman, R. A., Kraemer, F. B., McManaman, J. L., Obin, M. S., Puri, V., Yan, Q. W., Miyoshi, H. and Mashek, D. G. (2011). The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest 121(6): 2102-2110.
  2. Walther, T. C. and Farese, R. V., Jr. (2012). Lipid droplets and cellular lipid metabolism. Annu Rev Biochem 81: 687-714.

简介

脂滴(LD)是普遍存在的,动态的细胞器,并且作为中性脂质包括甘油三酯和胆固醇酯的储存库(Walther和Farese,2012)。 脂质物质进出LD的运动影响多种细胞过程,例如能量稳态,基于脂质的信号传导和膜内环境稳定(Greenberg等人,2011)。 例如,当增加脂质种类的合成或摄取时,中性脂质储存增强。 另一方面,细胞外信号可以增强包装在中性LD内的脂质物质的释放。 因此,涉及脂质代谢的主题的研究可能需要评估细胞中性脂质含量。 在该方案中,我们描述了荧光中性脂质染料4,4-二氟-1,3,5,7,8-五甲基-4-硼-3a,4a-二氮杂-s-引达省(BODIPY 493/503 ),以通过流式细胞术和通过显微镜观察LD来促进中性脂质含量的定量。

关键字:中性脂质, 脂滴, BODIPY

材料和试剂

  1. 35 mm细胞培养皿/孔
  2. 带有细胞过滤帽的流式细胞仪管(Corning,目录号:352235)
  3. 15 ml锥形管
  4. 35μm过滤器
  5. FACS管
  6. 玻璃幻灯片
  7. 圆盖玻片(12mm#1圆形)(VWR International,目录号:101415-528)
  8. 感兴趣的细胞系
    注意:对于该方案,我们利用A498透明细胞肾细胞癌细胞系(ATCC,目录号:A-498),但是该方法可以容易地用其他细胞系进行。
  9. 油酸(OA)(100mg/ml,含10%BSA的DPBS溶液)(Sigma-Aldrich,目录号:O3008)
  10. 将牛血清白蛋白(BSA)(在DPBS中10%,低内毒素,不含脂肪酸,适合于细胞培养,无菌过滤)(Sigma-Aldrich,目录号:A1595-50ML)
  11. 4,4-二氟-1,3,5,7,8-五甲基-4-硼-3a,4a-二氮杂-s-不对称 - 引达省(BODIPY 493/503)(Thermo Fisher Scientific,Molecular Probes< sup>,目录号:D3922)
  12. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  13. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco ,目录号:10010031)
  14. 含有酚红的胰蛋白酶-EDTA(0.25%)(Thermo Fisher Scientific,Gibco ,目录号:25200-056)
  15. HEPES
  16. NaCl
  17. CaCl 2
  18. 具有高葡萄糖和L-谷氨酰胺的Dulbecco改良的Eagle培养基(Thermo Fisher Scientific,Fisher Scientific,目录号:SH3024301)或适合于感兴趣的细胞系的其他培养基
  19. 胎牛血清(FBS)(Gemini Bio-Products,Foundation TM ,目录号:900-108)或适用于感兴趣的细胞系的其他FBS
    注意:对于该方案,细胞在DMEM + 10%FBS中生长。 
  20. 4%多聚甲醛(PFA)
  21. 具有4',6-二脒基-2-苯基吲哚(DAPI)(Thermo Fisher Scientific,Molecular Probes ,目录号:P36935)的Prolong Gold抗衰退试剂,
  22. 胶原I,大鼠尾(100mg)(Corning,目录号:354236)
  23. 2μMBODIPY染色溶液(见配方)
  24. 10x流式细胞仪缓冲液(见配方)
    注意:也可以购买10x缓冲液[10x膜联蛋白结合缓冲液(BD,目录号:556454)]。

设备

  1. 钳子
  2. 配备有488mm激光器和用于测量FITC或GFP(,533/30)的过滤器组的流式细胞仪
    注意:在本研究中,使用了BD Accuri C6仪器。

程序

  1. BODIPY染色流式细胞术
    1. 在与研究相关的培养条件下培养细胞。 35mm培养皿/孔对于该测定中所需的细胞数量是足够的。 对于我们的测定,在35mm孔中50,000个A498细胞就足够了
      1. 用30μM油酸过夜温育细胞可以作为增加中性脂质含量的阳性对照,因为油酸是甘油三酯合成和储存的有效诱导剂。 不含脂肪酸的BSA用作对照。
    2. 在感兴趣的时间点,制备2μMBODIPY染色溶液的PBS。 每个样品所需的染色溶液的体积对应于用于培养细胞的培养基的体积。
    3. 使用3ml PBS快速冲洗细胞以去除培养基/血清。
    4. 孵育在BODIPY染色溶液在黑暗中15分钟,在37℃。 包括流式细胞术的未染色对照。
      注意:从这一点,尽可能地保护样品免受光。
    5. 用3ml PBS快速冲洗细胞以去除染色溶液。
    6. 胰蛋白酶化细胞以产生单细胞悬浮液。 对于本方案中使用的A498细胞系,将细胞与胰蛋白酶-EDTA(0.25%)在37℃孵育5分钟。
    7. 加入5毫升PBS,并将细胞悬浮液转移到15毫升锥形管。
    8. 丸粒细胞在250×g下,5分钟,4℃。
    9. 吸出上清液,使用3ml PBS快速冲洗细胞沉淀,并在250×g,5分钟,4℃沉淀细胞。
    10. 小心吸出上清液,并悬浮细胞在300μl1x流式细胞仪缓冲液。
    11. 将细胞悬液通过35μm过滤器进入FACS管。
    12. 执行流式细胞术。 每个条件最少获得10,000个事件。
    13. 研究者可以将数据分析为平均荧光(图1A)或将数据显示为直方图(图1B)。

  2. BODIPY染色显微镜
    1. 高压灭菌在一个玻璃瓶的盖玻片。
    2. 在组织培养罩,将盖玻片放入35毫米细胞培养皿。
    3. 准备在PBS中的2mg/ml胶原溶液。
    4. 用胶原处理盖玻片以促进细胞粘附。 加入3毫升胶原溶液到培养皿中,在37℃孵育30分钟 注意:使用镊子确保盖玻片与培养皿底部齐平,消除盖玻片下可能有的气泡。
    5. 吸出胶原溶液。
    6. 用PBS洗涤。
    7. 向培养皿中加入PBS,并置于UV灯下的培养罩中灭菌。
    8. 平板细胞到含有盖玻片的培养皿中。在染色时应确定最佳细胞数目以达到30-50%的汇合,以允许适当的成像。对于在该方案中使用的A498细胞,将100,000个细胞接种在35mm孔中,以允许在接种后48小时染色。
    9. 在与您的实验相关的培养条件下孵育。
      1. 对于该方案,将A498细胞在补充有10%FBS的DMEM(高葡萄糖,L-谷氨酰胺,丙酮酸钠)中在37℃温育。
      2. 用30μM油酸与BSA一起温育细胞可以作为增加中性脂质含量的阳性对照,因为油酸是甘油三酯合成和储存的有效诱导剂。不含脂肪酸的BSA用作对照。
    10. 在感兴趣的时间点,在PBS中制备2μMBODIPY染色溶液。
      1. 对于该方案,在与BSA或BSA +油酸孵育过夜后,在铺板后48小时染色A498细胞。
    11. 用3ml PBS洗涤细胞。
    12. 在37℃下在3ml染色溶液上孵育15分钟 注意:从这一点,尽可能地保护样品免受光。
    13. 在3ml PBS中洗涤两次。
    14. 修复细胞在3毫升4%PFA在室温下30分钟。
    15. 去除4%PFA。
    16. 在PBS中洗涤样品3×5分钟。
    17. 使用镊子将盖玻片安装到载玻片上。
      1. 在玻片上加入一滴Prolong 金抗Dade试剂。
      2. 使用镊子拿起盖玻片并放置在安装溶液滴上,确保带有细胞的一侧面朝下放置在安装溶液上。
    18. 让安装溶液在室温下固化过夜。
    19. 载玻片可以储存在4°C或立即成像(图1C)。


      图1.细胞内中性LD的定量和可视化。 A。 A498细胞在BSA(0.2%)或30μM油酸与BSA的存在下生长过夜。根据标题为"BODIPY染色用于流式细胞术"的部分分析细胞。对于每个样品,将平均FL-1面积归一化为BSA处理的样品的平均FL-1面积。注意,油酸增加FL-1面积。 P - 值由非配对学生的测试决定。 *** P 0.001)。 B.(A)中描述的细胞的代表性直方图。 C.将生长在盖玻片上的A498细胞用BSA(0.2%)或30μM油酸与BSA处理过夜。根据标题为"BODIPY染色用于显微镜"的章节分析细胞。比例尺=5μm。

食谱

  1. 2μMBODIPY染色溶液
    1. 准备5 mM BODIPY储备液
      将1.3mg BODIPY溶解于1ml DMSO中,并可储存于-20℃。
    2. 2μMBODIPY染色溶液可以通过在PBS中稀释储备溶液1:2,500来制备。
  2. 10x流式细胞仪缓冲液
    0.1 M HEPES(pH 7.4)
    1.4 M NaCl
    25mM CaCl 2。 注意:
    1. 10x缓冲液可储存于4°C。
    2. 使用MilliQ水稀释至1x。

致谢

这项工作得到了NIH授予2-P01-CA104838到M.C.S和NIH授权5-F30-CA177106到B.Q.

参考文献

  1. Green,AS,Coleman,RA,Kraemer,FB,McManaman,JL,Obin,MS,Puri,V.,Yan,QW,Miyoshi,H.and Mashek,DG(2011)脂滴在啮齿动物和人的代谢疾病中的作用。 Clin Invest 121(6):2102-2110。
  2. Walther,TC和Farese,RV,Jr。(2012)。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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