搜索

Fat Turnover Assay in Drosophila
果蝇的脂肪转化测定   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Like all animals, Drosophila shows robust fat (triglyceride) turnover, i.e., they synthesize, store and utilize triglyceride for their daily metabolic needs. The protocol describes a simple assay to measure this turnover of triglycerides in Drosophila.

Background

Almost all animals store energy reserves in the form of glycogen and triglycerides. Many physiological, pathological and environmental conditions cause changes in the total level of these energy reserves, especially triglycerides. However, it’s not always clear whether the resulting changes in triglycerides are due to reduced breakdown, increased synthesis or vice versa. With this protocol, it is possible to determine both the rate of synthesis and degradation of the newly synthesized triglycerides in flies.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes
  2. Hamilton glass syringes (Hamilton, catalog number: Gastight 1700 )
  3. Metallic needle
  4. Razor blades (VWR, catalog number: 55411 )
  5. TLC silica gel 60 plates (Figure S1) (EMD Millipore, catalog number: 105626 )
  6. Drosophila vial (Genesee Scientific, Flystuff, catalog number: 32-109 )
  7. Whatman® chromatography paper (Sigma-Aldrich, catalog number: WHA3030861 )
  8. Drosophila melanogaster
  9. D-[14C(U)]-glucose (PerkinElmer, catalog number: NEC042V250UC )
  10. Yeast extract
  11. Sugar
  12. Liquid nitrogen and nitrogen gas
  13. Chloroform (Sigma-Aldrich, catalog number: 366927 )
  14. Methanol (EMD Millipore, catalog number: MX0475-1 )
  15. Cupric sulfate acid (EMD Millipore, catalog number: 102790 )
  16. O-phosphoric acid (85%) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A242-212 )
  17. Lipid standards:
    1. Triolein (Sigma-Aldrich, catalog number: T7140 )
    2. Phosphatidylcholine (Sigma-Aldrich, catalog number: P3556 )
    3. Phosphatidylinositol (Sigma-Aldrich, catalog number: P6636 )
    4. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
    5. Lauric acid (Sigma-Aldrich, catalog number: L556 )
    6. Myristic acid (Sigma-Aldrich, catalog number: M3128 )
    7. Palmitic acid (Sigma-Aldrich, catalog number: P0500 )
  18. Hexane (Sigma-Aldrich, catalog number: 32293 )
  19. Diethyl ether (Sigma-Aldrich, catalog number: 309966 )
  20. Acetic acid (AMRESCO, catalog number: 0714 )
  21. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  22. Scintillation fluid (PerkinElmer, catalog number: Ultima GoldTM/6013329 )
  23. Drosophila food recipes (see Recipes)
  24. Solvent mixture (see Recipes)
  25. Cupric sulfate/phosphoric acid solution (see Recipes)
  26. 0.9% NaCl solution (see Recipes)

Equipment

  1. TLC chamber (Clarkson Laboratory and Supply, model: Latch-Lid ChromatoTank 80-30 )
  2. Kontes microcentrifuge motor and pestles (Sigma-Aldrich, catalog number: Z359971-1EA )
  3. Centrifuge (Eppendorf, model: Centrifuge 5810R )
  4. Benchtop vacuum oven (VWR, model: 97027 )
  5. Scintillation counter (Beckman Coulter, model: LS6500 )
  6. Scintillation vials (PerkinElmer, catalog number: 6000292 )
  7. Nalgene PPCO wash bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2405-0500 )

Procedure

  1. The assay describes fat turnover in adult mated Drosophila females maintained on two different diets. The diets are HY (High yeast containing 5% yeast extract and 5% sugar) and LY (low yeast, containing 0.5% yeast extract and 5% sugar, see Katewa et al., 2016). On the tenth day about 300 flies (12 batches of 25 flies each) were transferred to fresh food vials with 2 µCi of 14C labeled glucose. Vials were prepared by adding 30 µl of 5% sugar/glucose solution on top and allowed to settle for 4 h. For preparing the sugar/glucose solution 100 µl of D-[14C(U)]-glucose was added to 900 µl of 5% sugar solution.
    Note: When you add 30 µl of sugar/glucose solution on top of the food, it’s best to use the sugar concentration that is closer to the concentration of sugar present in the regular lab fly food. For example, if your lab uses 10% sugar in the fly food, use 10% sugar solution to make the sugar/glucose solution.
  2. The flies were maintained on the labeled food for 24 h, after which half of the flies were snap-frozen in liquid nitrogen. This is the 0 h sample. The other half was transferred to a fresh non-radioactive food vial and was kept on this food for the next 60 h, and was then immediately frozen. This is the 60 h sample.
  3. For lipid extraction, weigh about 20 mg of flies/replicate. The weight is important for normalization and extraction of lipids. The number of flies will differ depending on the diets used in the experiment. For example, to get about 20 mg of total weight from the HY group, I need about 15-18 female flies, whereas for the LY group, I need about 22-25 flies. Homogenize the flies in about 100 µl of 0.9% NaCl in a 1.5 ml Eppendorf tube with a Kontes microcentrifuge motor and pestle. Transfer the homogenate to a 2 ml glass vial. Use additional 100 µl of 0.9% NaCl solution to rinse the Eppendorf tube and transfer to the glass vial. Add 800 µl of chloroform:methanol (2:1, v/v) to the glass vial with the homogenate, vortex for 15 sec and let it stand at room temperature for 20 min. Vortex again for 15 sec and centrifuge at 1,640 x g for 10 min to separate the two phases. By using a glass Hamilton syringe, carefully remove the lower phase containing the lipid fractions (Folch et al., 1957).
  4. The lipid fraction is transferred to a glass vial and dried under a continuous stream of nitrogen gas (always in a chemical fume hood that is designated for radioactive work). A glass pipette or a metallic needle could be attached at the end of gas supply and suspended in the glass vials (about one inch away from the solvent surface). Once completely dried, add 100 μl of chloroform to the tube.
    Note: There are two ways to separate the lipids into different fractions. One way is by using the solid phase extraction (SPE) tubes (for details see Katewa et al., 2012); another way is to use thin layer chromatography (TLC) plates (Chatterjee et al., 2014; Katewa et al., 2016). Here I am describing the TLC protocol as it is cost effective, simpler and also provides a visual separation of lipids.
  5. A glass Hamilton syringe is used to spot the resuspended lipid on TLC plates. 25 μg triolein was loaded to identify the triglyceride (TG) migration band. Plates are allowed to air dry in a chemical hood for about 20 min. Plates are developed in a solvent mixture (see Recipes section, Kishimoto et al., 2001) in a pre-conditioned TLC chamber. Pre-condition of the chamber involves few additional steps. The chamber is prepared by adding all the components of the solvent mix (Hexane/diethyl ether/acetic acid [70/30/1, v/v]) in the chamber, covering the chamber and mixing vigorously. Additionally, for proper migration of the solvent on the TLC, the inner side of the TLC chamber is lined with two overlapping sheets of Whatman® chromatography paper such that it lines the back and side walls of the chamber (Figure 1). Next, cover the chamber and let it equilibrate for 30 min, before you put the first TLC in the chamber.
    Note: Spots should be at least a centimeter above the solvent level.


    Figure 1. TLC chamber with lid. TLC chamber is lined with two overlapping sheets of Whatman® chromatography paper for saturation of the chamber. The inner Teflon® coated rack allows to simultaneous run several TLC plates.

  6. Load the dried TLC plate in the chamber and let it run for 10-15 min (depending on the rate of migration of the liquid front). Take the plates out and air dry in a chemical hood.
  7. For visual identification of TG spots, the TLC plate is sprayed with a solution of cupric sulfate/phosphoric acid (see Recipes section) and allowed to air dry for 30 min. The plates are heated in an oven at 125 °C for 30-45 min (until the TG band is visible). Remove the plate and let it cool. Figure 2 shows the efficiency of TLC to separate different lipid classes.


    Figure 2. TLC separation of different lipids from adult female Drosophila melanogaster. Total lipid was extracted from 15-20 adult female flies, dried and resuspended in 100 µl of chloroform and loaded on TLC plates. The plates were developed further to visualize migration of different lipids. Lane arrangements are as follows: Lane 1, Phospholipid (PL, a 1:1 mixture of 10 µg phosphatidylcholine and phosphatidylinositol). Lane 2, Triglyceride (TG, 25 µg triolein). Lane 3, Cholesterol (CHL, 10 µg). Lane 4, Lauric acid (LA, 10 µg). Lane 5, Myristic acid (MA, 10 µg). Lane 6, Palmitic acid (PA, 10 µg). Lane 7, ALL (All standards mixed in equal proportions) and Lane 8, 100 µl Fly sample.

  8. The TG bands from the plates are scraped with a razor blade and transferred to scintillation vials. Add about 0.5 ml of hexane to extract the TG from the silica. Add 3 ml of the scintillation fluid, mix and count the 14C radioactivity. Radioactivity count in the 0 h samples indicate the amount of incorporation of glucose into triglycerides and radioactivity count in 60 h samples indicate the amount of label TG still retained. The difference between 0 h and 60 h samples indicate the breakdown of the labeled triglycerides.

Data analysis

The amount of 14C present in 0 h sample denotes the de novo synthesis of TG and is expressed as CPM/mg fly weight. One experiment containing 5-6 independent samples of 15-25 flies is used to obtain mean ± SEM values. Additional experimental repeats should be carried out to confirm the observed results. Based on the design of the study, one can use Student’s t-test or ANOVA for obtaining the significance. Here, two different diets have been used, and the results (Figure 3) suggest that female flies on low yeast (LY) diet show increased de novo synthesis and a faster breakdown of TG when compared to the flies fed on high yeast (HY) diet.
Note: It’s best to use independent bottles to obtain flies for replicates in one experiment.


Figure 3. Fat turnover in adult female flies fed on high yeast (HY) and low yeast (LY) diets. The amount of 14C incorporation in 0 h sample indicates the de novo synthesis of TGs. The 60 h sample indicates the breakdown. Student’s t-test was used to measure statistical significance, and error bars denote SEM of five independent preparations (*indicates P < 0.05 and **indicates P < 0.001)

Recipes

  1. Drosophila food recipes
    Detailed fly media recipes can be found in the Supplemental Experimental Procedures section of Katewa et al. (2016).
  2. Solvent mixture
    Hexane/diethyl ether/acetic acid (70/30/1, v/v). Add 105 ml of hexane, 45 ml of diethyl ether and 1.5 ml of acetic acid in the TLC chamber and mix vigorously.
  3. Cupric sulfate/phosphoric acid solution
    8% cupric sulfate in 10% aqueous phosphoric acid
  4. 0.9% NaCl solution
    9 g NaCl in 1 L of double-distilled water

Acknowledgments

SDK acknowledges support from American Federation of Aging Research and Larry L. Hillblom Foundation grants.

References

  1. Chatterjee, D., Katewa, S. D., Qi, Y., Jackson, S. A., Kapahi, P., and Jasper, H. (2014). Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes. Proc Natl Acad Sci U S A 111(50): 17959-17964.
  2. Folch, J., Lees, M. and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226(1): 497-509.
  3. Katewa S.D, Akagi K, Bose N, Rakshit K, Camarella T, Zheng X, Hall D, Davies S, Nelson C, Brem RB, Ramanathan A, Sehgal A, Giebultowicz J.M and Kapahi P (2016). Peripheral circadian clocks mediate dietary restriction dependent changes in lifespan and fat metabolism in Drosophila. Cell Metab. 23(1):143-154.
  4. Katewa, S. D., Demontis, F., Kolipinski, M., Hubbard, A., Gill, M. S., Perrimon, N., Melov, S. and Kapahi, P. (2012). Intramyocellular fatty-acid metabolism plays a critical role in mediating responses to dietary restriction in Drosophila melanogaster. Cell Metab 16(1): 97-103.
  5. Kishimoto, K., Urade, R., Ogawa, T. and Moriyama, T. (2001). Nondestructive quantification of neutral lipids by thin-layer chromatography and laser-fluorescent scanning: suitable methods for "lipidome" analysis. Biochem Biophys Res Commun 281(3): 657-662.

简介

像所有动物一样,果蝇表现出强劲的脂肪(甘油三酯)周转,即,它们合成,储存和利用甘油三酯用于其日常代谢需要。 该方案描述了测量果蝇中甘油三酯转化的简单测定法。 几乎所有的动物都以 糖原和甘油三酯。 许多生理,病理和环境条件引起这些能量储备的总水平的变化,特别是甘油三酯。 然而,并不总是清楚甘油三酯中所导致的变化是由于减少的分解,增加的合成,或反之亦然。 使用此协议,可以确定新合成的甘油三酯在苍蝇中的合成速率和降解。

材料和试剂

  1. 1.5 ml Eppendorf管
  2. Hamilton玻璃注射器(Hamilton,目录号:Gastight 1700)
  3. 金属针
  4. 剃刀刀片(VWR,目录号:55411)
  5. TLC硅胶60板(图S1 < a)(EMD Millipore,目录号:105626)
  6. 果蝇(Genesee Scientific,Flystuff,目录号:32-109)
  7. Whatman色谱层析纸(Sigma-Aldrich,目录号:WHA3030861)
  8. 黑腹果蝇
  9. D - [14 C(U)] - 葡萄糖(PerkinElmer,目录号:NEC042V250UC)
  10. 酵母提取物

  11. 液氮和氮气
  12. 氯仿(Sigma-Aldrich,目录号:366927)
  13. 甲醇(EMD Millipore,目录号:MX0475-1)
  14. 硫酸铜(EMD Millipore,目录号:102790)
  15. O-磷酸(85%)(Thermo Fisher Scientific,Fisher Scientific,目录号:A242-212)
  16. 脂质标准:
    1. 三油酸甘油酯(Sigma-Aldrich,目录号:T7140)
    2. 磷脂酰胆碱(Sigma-Aldrich,目录号:P3556)
    3. 磷脂酰肌醇(Sigma-Aldrich,目录号:P6636)
    4. 胆固醇(Sigma-Aldrich,目录号:C8667)
    5. 月桂酸(Sigma-Aldrich,目录号:L556)
    6. 肉豆蔻酸(Sigma-Aldrich,目录号:M3128)
    7. 棕榈酸(Sigma-Aldrich,目录号:P0500)
  17. 己烷(Sigma-Aldrich,目录号:32293)
  18. 二乙醚(Sigma-Aldrich,目录号:309966)
  19. 乙酸(AMRESCO,目录号:0714)
  20. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  21. 闪烁液(PerkinElmer,目录号:Ultima Gold TM /6013329)
  22. 食谱(参见食谱)
  23. 溶剂混合物(参见配方)
  24. 硫酸铜/磷酸溶液(参见配方)
  25. 0.9%NaCl溶液(见配方)

设备

  1. TLC室(Clarkson Laboratory and Supply,型号:Latch-Lid ChromatoTank 80-30)
  2. Kontes微量离心机马达和杵(Sigma-Aldrich,目录号:Z359971-1EA)
  3. 离心机(Eppendorf,型号:Centrifuge 5810R)
  4. 台式真空炉(VWR,型号:97027)
  5. 闪烁计数器(Beckman Coulter,型号:LS6500)
  6. 闪烁瓶(PerkinElmer,目录号:6000292)
  7. Nalgene PPCO洗瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2405-0500)

程序

  1. 该测定描述了维持在两种不同饮食中的成年交配的果蝇雌性的脂肪周转。饮食是HY(含有5%酵母提取物和5%糖的高酵母)和LY(低酵母,含有0.5%酵母提取物和5%糖,参见Katewa等人,2016)。在第十天,将约300只苍蝇(12批,每只25只苍蝇)转移到具有2μCi14 C标记的葡萄糖的新鲜食品小瓶中。通过在顶部加入30μl的5%糖/葡萄糖溶液制备小瓶,并使其沉降4小时。为了制备糖/葡萄糖溶液,将100μl的D - [葡萄糖] 14 C(U)] - 葡萄糖加入到900μl的5%糖溶液中。
    注意:当在食物顶部添加30μl糖/葡萄糖溶液时,最好使用更接近常规实验室飞行食物中糖浓度的糖浓度。例如,如果您的实验室在飞行食物中使用10%的糖,则使用10%的糖溶液制作糖/葡萄糖溶液。
  2. 将苍蝇保持在标记的食物上24小时,之后将一半的苍蝇在液氮中速冻。这是0小时的样本。将另一半转移到新鲜的非放射性食品小瓶中,并在此食物上保持接下来的60小时,然后立即冷冻。这是60小时的样本。
  3. 对于脂质提取,称重约20mg苍蝇/复制。重量对于脂质的归一化和提取是重要的。果蝇的数量将根据实验中使用的饮食而不同。例如,要从HY组得到约20毫克的总重量,我需要约15-18雌性苍蝇,而对于LY组,我需要约22-25苍蝇。使用Kontes微量离心机和研杵在1.5ml Eppendorf管中将苍蝇在约100μl的0.9%NaCl中匀浆化。将匀浆转移到2ml玻璃小瓶中。使用额外的100μl的0.9%NaCl溶液冲洗Eppendorf管,并转移到玻璃小瓶。加入800微升氯仿:甲醇(2:1,v/v)与玻璃小瓶与匀浆,涡旋15秒,并让其在室温下静置20分钟。再次涡旋15秒并在1,640×g离心10分钟以分离两相。通过使用玻璃Hamilton注射器,小心地除去含有脂质级分的下相(Folch等人,1957)。
  4. 将脂质级分转移到玻璃小瓶中并在连续氮气流下干燥(总是在指定用于放射性工作的化学通风橱中)。玻璃吸管或金属针可以在气体供应的端部处附接并悬挂在玻璃小瓶中(离溶剂表面大约一英寸)。一旦完全干燥,加入100微升氯仿的管。
    注意:有两种方法将脂质分成不同的级分。一种方法是使用固相萃取(SPE)管(详见Katewa等人,2012);另一种方法是使用薄层色谱(TLC)板(Chatterjee et al。,2014; Katewa et al。,2016)。这里我描述TLC协议,因为它是有成本效益的,更简单,也提供脂质的视觉分离。
  5. 使用玻璃Hamilton注射器在TLC板上点样重悬的脂质。加载25μg甘油三油酸酯以鉴定甘油三酯(TG)迁移带。将板在化学罩中风干约20分钟。在预处理的TLC室中,在溶剂混合物(参见Recipe section,Kishimoto等人,2001)中显影板。腔室的前提条件包括几个额外的步骤。通过将室中的溶剂混合物(己烷/乙醚/乙酸[70/30/1,v/v])的所有组分加入,覆盖室并剧烈混合来制备室。另外,为了使溶剂在TLC上正确迁移,TLC室的内侧衬有两层重叠的Whatman色谱纸,使得它排列室的背面和侧壁图1)。接下来,盖上腔室,让它平衡30分钟,然后将第一个TLC放入腔室 注意:斑点应至少比溶剂水平高出一厘米。


    图1.具有盖的TLC室。 TLC室内衬有两个重叠的Whatman色谱纸,用于室的饱和。内部Teflon ?涂层机架允许同时运行多个TLC板
  6. 将干燥的TLC板装载在室中,并使其运行10-15分钟(取决于液体前沿的迁移速率)。取出板,在化学罩中风干。
  7. 为了目测TG斑点,用硫酸铜/磷酸溶液喷雾TLC板(参见食谱部分),并使其风干30分钟。将板在烘箱中在125℃加热30-45分钟(直到TG带可见)。取出板,让它冷却。图2显示了TLC分离不同脂质类别的效率

    图2.不同脂质与成年雌性果蝇黑腹果蝇的TLC分离从15-20只成年雌蝇中提取总脂质,干燥并重悬于100μl氯仿中并加载在TLC板上。进一步显影板以显现不同脂质的迁移。泳道布置如下:泳道1,磷脂(PL,10μg磷脂酰胆碱和磷脂酰肌醇的1:1混合物)。泳道2,甘油三酯(TG,25μg甘油三油酸酯)。泳道3,胆固醇(CHL,10μg)。泳道4,月桂酸(LA,10μg)。泳道5,肉豆蔻酸(MA,10μg)。泳道6,棕榈酸(PA,10μg)。泳道7,ALL(所有标准以相等比例混合)和泳道8,100μl飞样品
  8. 来自板的TG带用剃刀刀片刮下并转移到闪烁瓶中。加入约0.5ml己烷以从二氧化硅中提取TG。加入3ml闪烁液,混合并计数 14℃放射性。 0小时样品中的放射性计数表示葡萄糖掺入甘油三酯的量,并且在60小时样品中的放射性计数表示仍保留的标记TG的量。 0h和60h样品之间的差异表明标记的甘油三酯的分解

数据分析

存在于0小时样品中的14 C的量表示TG的新合成,并表示为CPM/mg飞重。使用包含5-6个独立的15-25个蝇的样本的一个实验来获得平均值±SEM。应进行额外的实验重复以确认观察结果。基于研究的设计,可以使用Student's - 检验或ANOVA获得显着性。在这里,已经使用了两种不同的饮食,并且结果(图3)表明,与苍蝇相比,低酵母(LY)饮食中的雌蝇显示增加的新合成和更快的TG分解喂食高酵母(HY)饮食 注意:最好使用独立瓶子在一个实验中获得飞行副本。


图3.在高酵母(HY)和低酵母(LY)饮食饲喂的成年雌性果蝇中的脂肪周转。在0小时样品中掺入的 14的量表示 TG的新合成。 60小时样品指示击穿。 Student's t检验用于测量统计学显着性,误差条表示5种独立制剂的SEM(*表示P <0.05,**表示P <0.001)

食谱

  1. 果蝇食谱
    详细的飞行培养基配方可以在Katewa等人的补充实验程序部分中找到。 (2016)。
  2. 溶剂混合物
    己烷/乙醚/乙酸(70/30/1,v/v)。在TLC室中加入105ml己烷,45ml乙醚和1.5ml乙酸,并剧烈搅拌。
  3. 硫酸铜/磷酸溶液
    8%硫酸铜在10%磷酸水溶液中
  4. 0.9%NaCl溶液
    9克NaCl在1升双蒸水中洗涤

致谢

SDK承认美国老龄化研究联合会和Larry L. Hillblom基金会的资助。

参考文献

  1. Chatterjee,D.,Katewa,SD,Qi,Y.,Jackson,SA,Kapahi,P.,and Jasper,H。(2014)。  通过dILP6诱导的胰岛素信号在果蝇细胞中控制对空腹的代谢适应。 Proc Natl Acad Sci USA 111(50):17959-17964。
  2. Folch,J.,Lees,M.和Sloane Stanley,GH(1957)。  一种用于从动物组织中分离和纯化总脂质的简单方法。 J Biol Chem 226(1):497-509。
  3. Katewa SD,Akagi K,Bose N,Rakshit K,Camarella T,Zheng X,Hall D,Davies S,Nelson C,Brem RB,Ramanathan A,Sehgal A,Giebultowicz JM and Kapahi P(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26626459"target ="_ blank">外周昼夜节律调节膳食限制依赖于寿命和脂肪代谢的变化,果蝇。 细胞代谢。 23(1):143-154
  4. Katewa,SD,Demontis,F.,Kolipinski,M.,Hubbard,A.,Gill,MS,Perrimon,N.,Melov,S。和Kapahi,P。(2012)。肌内脂肪酸代谢在介导对果蝇中饮食限制的反应中起关键作用/em> melanogaster。 Cell Metab 16(1):97-103。
  5. Kishimoto,K.,Urade,R.,Ogawa,T.and Moriyama,T。(2001)。  通过薄层色谱和激光荧光扫描对中性脂质的非破坏性定量:用于"脂质体"分析的合适方法。生物化学与生物物理学通讯281(3):657-662。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Katewa, S. D. (2016). Fat Turnover Assay in Drosophila. Bio-protocol 6(21): e1996. DOI: 10.21769/BioProtoc.1996.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。