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Jan 2020

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Triacylglycerol Measurement in HeLa Cells
HeLa细胞中三酰甘油的测定   

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Abstract

Lipid droplets store triacylglycerols (triglycerides) and sterol esters to regulate lipid and energy homeostasis. Triacylglycerol measurement is often performed during the investigation of lipid droplet formation and growth. This protocol describes a reliable method using a fluorometric lipid quantification kit to measure triacylglycerols extracted from HeLa cells, which were treated with oleic acid to trigger the formation of lipid droplets. The lipid quantification kit employs a lipid-binding molecule that emits bright fluorescence only when bound to extracted triacylglycerols, whose content can be quantified by a simple fluorescence readout.

Keywords: Lipid droplet (脂滴), Triacylglycerol (三酰甘油), Triglyceride (甘油三酯), Lipid extraction (油脂提取), Phospholipid (磷脂), Sterol ester (甾醇酯), Oleic acid (油酸), Oleate (油酸盐)

Background

Lipid droplets (LDs) are unique organelles with characteristic functions. Different from common cellular organelles, each LD is defined by a monolayer of phospholipids, which encloses a core of neutral lipids such as triacylglycerol (TAG), also known as triglyceride, and sterol esters. As storage organelles, LDs play a major role in the homeostasis of lipids and energy, as well as other cellular events. By storing excess fatty acids in the form of TAG, LDs prevent lipotoxicity and protect cells against oxidative stress. Importantly, LDs make contacts with virtually all other organelles and regulate critical cellular processes, including membrane trafficking, protein turnover and viral infection (Olzmann and Carvalho, 2018; Gao et al., 2019). Moreover, the LD surface is decorated with various proteins that are involved in the synthesis and hydrolysis of TAG, and in the transport of lipids between LDs and other organelles (Kory et al., 2016; Kumar et al., 2018; Du et al., 2020).

Eukaryotic cells have well-established pathways to synthesize TAG stored in LDs (Coleman and Mashek, 2011). Studies on the formation and growth of LDs often involve the extraction of TAG from cells or tissues for quantification. Some sensitive methods, such as LC-MS, are available for this purpose, but requiring expensive equipment and being time-consuming. Commercially available lipid quantification kit, on the other hand, can provide a quick, accurate, and economical method to measure cellular levels of TAG. In this protocol, we describe a reliable method using a fluorometric lipid quantification kit to measure TAG extracted from HeLa cells that were grown in the presence of oleic acid to induce LD formation. The lipid quantification kit takes advantage of a lipid binding molecule that emits bright fluorescence only when bound to extracted TAG. Thus, the amount of TAG can be readily quantified by a simple fluorescence readout.

Materials and Reagents

  1. Cell culture dish (60 x 15 mm) (Corning, catalog number: CLSE430166)

  2. 2 ml glass vial (Agilent, catalog number: 5190-9062)

  3. Glass bottle, 100 ml (Therfmo Fisher Scientific, catalog number: FSBFB33144)

  4. Microplates for Fluorescence-based Assays, 96-well (Thermo Fisher Scientific, catalog number: M33089)

  5. 1.5 ml microcentrifuge tubes (Sarstedt, catalog number: 72.690.001)

  6. Serological pipettes, 5 ml, 10 ml, and 25 ml (Corning, catalog numbers: CLS4487, 4488, 4489)

  7. Pipette tips with filter (Interpath Services, catalog numbers: 24750, 24900)

  8. Falcon tubes, 15 ml, 25 ml (Corning, catalog numbers: CLS430829, 430791)

  9. HeLa cell line (ATCC CCL-2TM)

  10. Fluorometric Lipid Quantification Kit (Cell Biolabs, catalog number: STA-617)

  11. Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, catalog number: 11995073)

  12. Fetal bovine serum (FBS) (Bovogen, catalog number: FFBS-500)

  13. Penicillin-Streptomycin-Glutamine (100x) (Gibco, catalog number: 10378016)

  14. DPBS, no calcium, no magnesium (Gibco, catalog number: 14190250)

  15. Oleic Acid-Albumin from bovine serum (Sigma-Aldrich, catalog number: O3008-5ML)

  16. Methanol (Univar, catalog number: AJA2314-2.5LGL)

  17. Hexane (VWR International, catalog number: VWRC24577.323)

  18. Chloroform (Chem-supply Pty Ltd Australia, catalog number: RP1027E-G2.5L)

  19. Isopropanol (Unichrom, catalog number: 2323-2.5GL)

  20. Sodium hydroxide (Univar, catalog number: AJA482-500G)

  21. Bicinchoninic Acid Kit for protein determination (Sigma-Aldrich, catalog number: BCA1-1KT)

  22. Sodium chloride (Chem-Supply, catalog number: SA046-500G)

  23. TRIS (hydroxymethyl) aminomethane (Univar, catalog number: 1.08382.0500)

  24. Bovine serum albumin (Sigma-Aldrich, catalog number: A1933)

  25. Buffer A (see Recipes)

  26. Buffer B (see Recipes)

Equipment

  1. Biological Safety Cabinets (NuAire, model: 5437-400E)

  2. CO2 incubator (Binder, model: CB 150)

  3. Fume cupboard (Dynaflow, model: FC1800)

  4. Heating block (Labnet International, model: D1100)

  5. Vortex mixer (Ratek, model: IC-VM1)

  6. POLARstar Omega Microplate Reader (BMG Labtech, catalog number: 415-0704)

  7. Water bath (Ratek, model: IC-WB1200D)

Procedure

  1. Cell culture

    1. Seed HeLa cells in 6-cm dishes (5 x 105 cells per dish) in 3 ml of DMEM supplemented with 10% FBS and 1x Penicillin-Streptomycin.

    2. Incubate cells at 37 °C in a humidified incubator with 5% CO2 for 24 h.

    3. Treat cells with 400 µM of oleic acid-albumin from bovine serum by adding 0.24 ml of the stock solution (3.3 mM) to 1.76 ml of culture medium.


  2. Lipid extraction

    1. Wash cells in the dishes twice with 2 ml of buffer A.

    2. Wash cells once with buffer B.

    3. Aspirate the buffer residues with pipette tips.

    4. Add 2 ml of hexane-isopropanol (3:2) to each dish and incubate for 30 min at room temperature in a fume hood with the lid on.

      Note: Hexane and chloroform are hazardous to breathe. Therefore, the handling of these reagents should be carried out in chemical fume hood that is safely vented to outside the laboratory.

    5. Transfer the organic solvent from each dish to a 2 ml glass vial.

    6. Rinse each dish briefly with 1 ml of the same solvent and combine into the same glass vial. Carefully remove all of the organic solvents and save the dish for protein determination.

    7. Evaporate the solvent to dryness inside the fume hood by leaving the vial uncapped in the fume hood with the airflow running (takes approximately 16 h).


  3. Protein determination

    1. Add 1 ml of 0.1 M NaOH in water to the original cell culture dish and dissolve the cell remnants by pipetting up and down.

    2. Remove 20 µl of aliquots in duplicate and dilute in 20 µl of water for BCA assay to determine total proteins in each dish (protein amount of each assay x 50). A typical protein yield per dish is 500-600 µg.


  4. Lipid quantification with the Kit #STA-617 from Cell BioLabs with modifications

    1. Mix 50 ml of methanol with 25 ml of chloroform in a 100 ml glass bottle. Store at room temperature.

    2. Thoroughly resuspend each extracted TAG sample from Step B7 with 200 µl of methanol/chloroform mixture in capped glass vials using a vortex mixer at high speed for 30 s. Keep on ice after the vortexing.

    3. Prepare serial dilutions of lipid standards in the methanol/chloroform mixture, with a concentration range from 0 to 5 µg/µl (Table 1). Mix each standard well before further dilution.


      Table 1. Lipid standards with a serial dilution


    4. Add 40 µl of standards or TAG samples in duplicates to a 96-well microplate for fluorescence-based assays.

    5. Evaporate the organic solvent by incubating the plate on top of a heating block set as 55 °C for 30 min or until dryness in a safety chemical hood.

    6. Warm the 100x Fluorometric Reagent stock in a 37 °C water bath until thawed.

    7. Prepare 1x Fluorometric Reagent by adding 0.5 ml of the thawed 100x stock to 49.5 ml of water in a 50 ml falcon tube.

    8. Cool the 96-well plate on ice for 2 min.

    9. Add 40 µl of isopropanol to standards or TAG samples. Pipette up and down gently to mix each well.

    10. Add 200 µl of 1x Fluorometric Reagent to each well.

    11. Incubate the plate in the dark for 10 min at room temperature.

    12. Read the plate at 490 nm excitation and 585 nm emission with a POLARstar Omega Microplate Reader.

Data analysis

  1. Generate the standard curve by plotting the amount of lipid standards against the relative fluorescent unit (RFU). An example of the standard curve with an equation is shown in Figure 1.



    Figure 1. Fluorometric standard curve for TAG quantification


  2. Calculate the quantity of TAG in each sample well of the 96-well plate using the equation shown in the graph of the standard curve (Figure 1).

  3. Calculate the total quantity of TAG from each sample by multiplying the amount of TAG in the sample well with five (because 40 μl out of a 200 μl was measured per well). Typical yields of TAGs and protein per 6-cm dish of HeLa cells (~90% confluency) are shown in Table 2.


    Table 2. Yields of TAGs and proteins from each dish of HeLa cells


  4. Normalize the total quantity of TAG with the total amount of proteins determined in Procedure C (Table 2). An example of final results is shown in Figure 2.



    Figure 2. TAG quantities in HeLa cells (control vs. sample#1) determined by the fluorometric assay

Recipes

  1. Buffer A

    150 mM NaCl

    50 mM Tris-HCl, pH 7.4

    2 mg/ml Bovine serum albumin

  2. Buffer B

    150 mM NaCl

    50 mM Tris-HCl, pH 7.4

Acknowledgments

This work was supported by project grants from the National Health and Medical Research Council of Australia (APP1041301, 1141939, and 114472).

TAG extraction procedure described in this protocol was adapted from a well-defined early method ( Goldstein et al., 1983 ).

Competing interests

The authors declare no competing interests.

References

  1. Coleman, R. A. and Mashek, D. G. (2011). Mammalian Triacylglycerol Metabolism: Synthesis, Lipolysis, and Signaling. Chem Rev 111(10): 6359-6386.
  2. Du, X., Zhou, L., Aw, Y. C., Mak, H. Y., Xu, Y., Rae, J., Wang, W., Zadoorian, A., Hancock, S. E., Osborne, B., Chen, X., Wu, J. W., Turner, N., Parton, R. G., Li, P. and Yang, H. (2020). ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets. J Cell Biol 219(1).
  3. Gao, M., Huang, X., Song, B. L. and Yang, H. (2019). The biogenesis of lipid droplets: Lipids take center stage. Prog Lipid Res 75: 100989.
  4. Goldstein, J. L., Basu, S. K. and Brown, M. S. (1983). Methods in Enzymology. Sciencedirect 98: 241-260.
  5. Kory, N., Farese, R. V. and Walther, T. C. (2016). Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets. Trends Cell Biology 26(7): 535-546.
  6. Kumar, N., Leonzino, M., Hancock-Cerutti, W., Horenkamp, F. A., Li, P., Lees, J. A., Wheeler, H., Reinisch, K. M. and Camilli, P. D. (2018). VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. J Cell Biol 217(10): 3625-3639.
  7. Olzmann, J. A. and Carvalho, P. (2018). Dynamics and functions of lipid droplets. Nat Rev Mol Cell Biol 20(3): 1.

简介

[摘要]脂滴商店吨riacylglycerols (甘油三酯)和甾醇酯调节脂质和能量稳态。通常在研究脂质滴的形成和生长过程中进行三酰基甘油的测量。该协议描述了一种使用荧光脂质定量试剂盒测量从HeLa细胞提取的三酰基甘油的可靠方法,该甘油经油酸处理后触发了脂质滴的形成。脂质定量试剂盒采用的脂质结合分子仅在与提取的三酰基甘油结合时才发出明亮的荧光,其含量可以通过简单的荧光读数来定量。

[背景]脂滴(LD)的是具有特征功能独特的细胞器。与常见的细胞器不同,每个LD由磷脂的单层定义,该磷脂包裹着中性脂质(如三酰甘油(TAG),也称为甘油三酸酯)和甾醇酯的核心。作为存储细胞器,LDs在脂质和能量的动态平衡以及其他细胞事件中起主要作用。通过以TAG的形式存储过量的脂肪酸,LDs可以防止脂毒性并保护细胞免受氧化应激。重要的是,LDs几乎与所有其他细胞器接触并调节关键的细胞过程,包括膜运输,蛋白质更新和病毒感染(Olzmann和Carvalho,2018; Gao等,2019)。此外,LD表面装饰有各种蛋白质,这些蛋白质参与TAG的合成和水解以及LD与其他细胞器之间的脂质运输(Kory等,2016; Kumar等,2018; Du等。 。,2020)。

真核细胞具有完善的途径来合成LDs中存储的TAG (Coleman和Mashek,2011)。关于LD的形成和生长的研究通常涉及从细胞或组织中提取TAG以进行定量。一些敏感的方法(例如LC-MS)可用于此目的,但需要昂贵的设备并且很费时间。另一方面,可商购的脂质定量试剂盒可提供一种快速,准确且经济的方法来测量TAG的细胞水平。在此协议中,我们描述了一种使用荧光脂质定量试剂盒来测量从HeLa细胞中提取的TAG的可靠方法,该细胞在油酸存在下生长以诱导LD形成。脂质定量试剂盒利用了脂质结合分子的优势,该分子仅在与提取的TAG结合后才发出明亮的荧光。因此,可以通过简单的荧光读数容易地定量TAG的量。

关键字:脂滴, 三酰甘油, 甘油三酯, 油脂提取, 磷脂, 甾醇酯, 油酸, 油酸盐

 
材料和试剂
 
1.细胞培养皿(60 x 15毫米)(Corning,目录号:CLSE430166)      
2. 2毫升玻璃小瓶(安捷伦,目录号:5190-9062)      
3. 100毫升玻璃瓶(Therfmo Fisher Scientific,目录号:FSBFB33144)      
4. 96孔荧光定量微孔板(Thermo Fisher Scientific,目录号:M33089)      
5. 1.5 ml微量离心管(Sarstedt ,目录号:72.690.001)      
6. 5 ml,10 ml和25 ml血清移液管(Corning,目录号:CLS4487、4488、4489)      
7.带过滤器的移液器吸头(Interpath Services,目录号:24750、24900)      
8.猎鹰管,15 ml,25 ml(Corning,目录号:CLS430829,430791)      
9. HeLa细胞系(ATCC CCL-2 TM )      
10.荧光脂质定量试剂盒(Cell Biolabs ,Inc.,目录号:STA-617)   
11. Dulbecco的改良Eagle's Medium(DMEM)(Gibco ,目录号:11995073)   
12.胎牛血清(FBS)(Bovogen ,目录号:FFBS-500)   
13.青霉素-链霉素-谷氨酰胺(100 x )(Gibco ,目录号:10378016)   
14. DPBS,无钙,无镁(Gibco ,目录号:14190250)   
15.来自牛血清的油酸-白蛋白(Sigma-Aldrich,目录号:O3008-5ML)   
16.甲醇(Univar,目录号:AJA2314-2.5LGL)   
17.己烷(VWR International,目录号:VWRC24577.323)   
18.氯仿(澳大利亚Chem -supply Pty Ltd,目录号:RP1027E-G2.5L)   
19.异丙醇(Unichrom ,目录号:2323-2.5GL)   
20.氢氧化钠(Univar,目录号:AJA482-500G)   
21.用于蛋白质测定的双辛可宁酸试剂盒(Sigma-Aldrich,目录号:BCA1-1KT)   
22.氯化钠(Chem- Supply,目录号:SA046-500G)   
23. TRIS(羟甲基)氨基甲烷(Univar的,目录号:1.08382.0500)   
24.牛血清白蛋白(Sigma-Aldrich,目录号:A1933)   
25.缓冲区A(请参阅食谱)   
26.缓冲区B(请参阅食谱)   
 
设备
 
生物安全柜(NuAire ,型号:5437-400E)
CO 2培养箱(粘合剂,型号:CB 150)
通风柜(Dynaflow ,型号:FC1800 )
加热块(Labnet International,型号:D1100)
涡旋混合器(Ratek ,型号:IC-VM1)
POLARstar Omega酶标仪(BMG Labtech ,目录号:415-0704)
水浴锅(Ratek ,型号:IC-WB1200D)
程序
 
细胞培养
将HeLa细胞接种在6厘米培养皿中(每个培养皿5 x 10 5个细胞),放入3 ml的DMEM中,添加10%FBS和1 x青霉素-链霉素。              
在37孵育细胞 在5%的CO 2加湿培养箱中于24 °C下放置24 ℃。
通过将0.24 ml的原液(3.3 mM )添加到1.76 ml的培养基中,用来自牛血清的400 µM的油酸-白蛋白处理细胞。
 
脂质提取
用2 ml缓冲液A清洗培养皿中的细胞两次。
用缓冲液B洗涤细胞一次。
用移液器吸头吸去缓冲液残留物。
向每个培养皿中加入2 ml的己烷-异丙醇(3:2),并在带盖的通风橱中于室温下孵育30分钟。
注意:己烷和氯仿对呼吸有害。因此,这些试剂的处理应在安全通风到实验室外部的化学通风橱中进行。
将每个皿中的有机溶剂转移到2 ml玻璃小瓶中。
用1毫升相同的溶剂短暂冲洗每个培养皿,然后合并到同一玻璃瓶中。小心除去所有有机溶剂,并保存培养皿以进行蛋白质测定。
通过使小瓶在通风橱中保持不开盖的状态,将溶剂蒸发到通风橱中干燥(大约需要16小时)。
 
蛋白质测定
向原始细胞培养皿中加入1 ml的0.1 M NaOH水溶液,并通过上下吹打溶解细胞残余物。
取出一式两份的20 µl等分试样,并在20 µl水中稀释以进行BCA测定,以确定每个培养皿中的总蛋白质(每次测定的蛋白质量x 50)。每盘的典型的蛋白产量为500 - 600微克。
 
使用Cell BioLabs的试剂盒#STA-617进行脂质定量和改进
在100 ml玻璃瓶中将50 ml甲醇与25 ml氯仿混合。存放在室温下。
彻底重悬从每个所提取的TAG样品步骤B7,在使用涡旋混合器以高速进行30秒加盖玻璃小瓶加入200μl甲醇/氯仿混合物中。涡旋后放在冰上。
在甲醇/氯仿混合物中制备脂质标准品的系列稀释液,浓度范围为0至5 µg / µl(表1)。在进一步稀释之前,将每个标准品充分混合。
表1.连续稀释的脂质标准品
 
管号
甲醇/氯仿混合物(微升)
脂质标准
(微升)
最终浓度
(微克/微升)
1个
390
10微升x 200微克/微升
5
2
200
200#1
2.5
3
200
200名#2
1.25
4
200
200名#3
0.625
5
200
200名#4
0.3125
6
200
第5名的200名
0.15625
7
200
第6名的200名
0.078125
8
200
0
0
 
将一式两份的40 µl标准品或TAG样品加至96孔微孔板中,以进行基于荧光的测定。
通过将板在设定为55 °C的加热块顶部温育30分钟或直至在安全化学通风橱中变干来蒸发有机溶剂。
在37 °C水浴中加热100 x荧光试剂储液直至融化。
通过在50 ml猎鹰管中将0.5 ml解冻的100 x原液添加到49.5 ml水中,制备1 x荧光试剂。
将96孔板在冰上冷却2分钟。
将40 µl异丙醇加入标准品或TAG样品中。轻轻地上下移液,以混合各孔。
向每个孔中加入200 µl 1 x荧光试剂。
在室温下于黑暗中孵育平板10分钟。
用POLARstar Omega微孔板读板器在490 nm激发和585 nm发射下读取板。
 
数据分析
 
通过相对于相对荧光单位(RFU)绘制脂质标准液的量来生成标准曲线。带有方程式的标准曲线示例如图1所示。


图URE 1.荧光为TAG定量标准曲线
 
使用标准曲线图(图1)中所示的公式计算96孔板每个样品孔中TAG的数量。
通过在样品中有五个TAG的量乘以以及计算TAG的从每个样品的总量(因为40微升出200微升每测定孔)。表2显示了每6厘米培养皿中HeLa细胞的TAGs和蛋白质的典型产量(约90%融合)。
 
表2.每道HeLa细胞培养皿中TAG和蛋白的产量
 
TAG(微克)
总蛋白(毫克)
TAG /蛋白质(µg / mg)
控制
 
 
 
14.361
0.530319
27.07994
15.739
0.602128
26.13897
15.027
0.580515
25.88562
16.311
0.562389
29.00307
11.937
0.487094
24.50657
17.485
0.574241
30.4489
 
 
 
 
样品#1
 
 
 
55.519
0.634895
87.44597
39.329
0.616071
63.8384
37.483
0.590276
63.50083
41.503
0.586093
70.81303
38.221
0.552628
69.16222
41.447
0.581212
71.31127
 
用步骤C中确定的蛋白质总量对TAG总量进行标准化(表2)。甲的最终结果N实施例示于图2。
 


图URE在HeLa细胞中TAG 2量(控制组。样品#1)确定由所述荧光测定法
 
菜谱
 
缓冲液A
150毫米氯化钠
50 mM的Tris-HCl ,pH 7.4
2 mg / ml牛血清白蛋白
缓冲液B
150毫米氯化钠
50 mM的Tris-HCl ,pH 7.4
 
致谢
 
这项工作得到了澳大利亚国家卫生与医学研究委员会(APP1041301、1141939和114472)的项目拨款的支持。
该协议中描述的TAG提取程序改编自定义明确的早期方法(Goldstein等,1983)。
 
利益争夺
 
作者宣称没有利益冲突。
 
参考文献
 
Coleman,RA和Mashek,DG(2011)。哺乳动物三酰基甘油代谢:合成,脂解和信号传导。Chem Rev 111(10):6359-6386。              
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Du, X. and Yang, H. (2020). Triacylglycerol Measurement in HeLa Cells. Bio-protocol 10(24): e3852. DOI: 10.21769/BioProtoc.3852.
  2. Du, X., Zhou, L., Aw, Y. C., Mak, H. Y., Xu, Y., Rae, J., Wang, W., Zadoorian, A., Hancock, S. E., Osborne, B., Chen, X., Wu, J. W., Turner, N., Parton, R. G., Li, P. and Yang, H. (2020). ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets. J Cell Biol 219(1).
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