参见作者原研究论文

本实验方案简略版
Nov 2014

本文章节


 

Ecdysone Quantification from Whole Body Samples of Drosophila melanogaster Larvae
黑腹果蝇幼虫全身蜕皮激素的定量分析   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass in insects, and the duration of this growth period is driven by a sequence of small ecdysone pulses that ultimately induce metamorphosis. Historically the biologically active form of ecdysone, 20-hydroxyecdysone (20E), was quantified using radio-immunoassays, bioassays, or chromatography assays. However, these assays are methodologically complicated and often time consuming. Furthermore, collecting samples for precise measurements of ecdysone concentrations using these assays is limited in small insects like Drosophila melanogaster. Here, we describe an accurate and sensitive method to collect carefully-staged third instar larvae suitable for preparing samples for ecdysone quantification using a commercially-available 20E enzyme immunoassay (EIA). Because we resynchronize larval development at the molt to the final instar, collect large samples, and weigh each sample, we are able to detect a small ecdysone peak early in the final instar known as the critical weight ecdysone peak. This method detects peaks as low as 6 pg 20E/mg larval sample, allowing us to quantify other small ecdysone peaks in flies – the necessary prerequisite for eventually determining their regulation and function.

Keywords: 20-hydroxyecdysone (20E) (20-羟基蜕皮酮), Larval synchronization (幼虫同步), Larval staging (幼虫分期), Enzyme immunoassay (EIA) (酶免疫分析), Drosophila melanogaster (黑腹果蝇), Whole body ecdysone quantification (全身脱皮激素定量), Critical weight (临界体重)

Background

In a broad range of animals, development is tightly regulated by steroid hormones. In mammals, the onset of puberty is primarily regulated by sex steroid hormones (Koyama et al., 2020). In insects, the steroid hormone ecdysone regulates the timing of molting, including the timing of the metamorphic molts. Because small fluctuations in the concentrations of ecdysone are predicted to trigger key developmental events, developing methods to detect changes in ecdysone concentrations is crucial for scientists aiming to evaluate steroid hormone function. In this protocol, we describe how to collect high-quality samples from developmentally synchronized larvae, and how to prepare these samples for ecdysone quantifications using a commercially-available enzyme immunoassay kit.


Materials and Reagents

  1. 1.5 ml microcentrifuge tubes

  2. Paper towels

  3. Parafilm (Bemis Company, PM996)

  4. Aluminum foil

  5. Disposable pestles

  6. Crystal cuvette

  7. 200 parental virgin female Drosophila melanogaster and 150 parental males

  8. Precoated (Mouse Anti-Rabbit IgG) ELISA 96-Well Strip Plate (Cayman Chemicals, catalog number: 400005), store at 4 °C

    We have successfully used these plates up to one year after purchase.

  9. Absolute methanol (VWR, Methanol absolute ≥ 99.8% ACS, catalog number: M1240-4LTGL), store at room temperature

  10. Absolute ethanol (VWR, Ethanol absolute ≥ 99.8%, Electran Molecular biology grade, catalog number: 437435L), store at room temperature

  11. 20-Hydroxyecdysone EIA Kit (Cayman Chemicals, catalog number: 501390)

  12. 20-Hydroxyecdysone (SciTech Chemicals, catalog number: S3314-001)

  13. Sucrose (50 g sucrose in 250 ml ddH2O. Preferably freshly prepared. Otherwise store at 4 °C for up to a week. Warm up to be about 20-25 °C before use)

  14. Materials need for fly husbandry (dry yeast, plastic disposable vials, plugs, 60 mm tissue culture plates, embryo collection chambers)

  15. Double-distilled water

  16. Dry ice

  17. 70% ethanol

  18. Molasses

  19. Sugar

  20. Cornmeal

  21. Yeast Extract

  22. Agar

  23. Cornmeal/molasses medium (fly medium, see Recipes)

Equipment

  1. Dumont #3 Forceps (Fine Science Tools, catalog number: 11231-30) or entomology forceps (Fisher Scientific, catalog number: S72110)

  2. Ultra-microbalance (Sartorius, Sartorius Cubis Series, SE2)

  3. Cordless pestle motor (VWR International, VWR Cordless Pestle Motor, catalog number: 47747-370)

  4. Microcentrifuge with refrigeration

  5. SpeedVac (Savant, SpeedVac Plus SC10A, catalog number: SC110A-8E210146-4K)

  6. Microplate reader, Victor3 (PerkinElmer, Victor3, catalog number: 1420-050)

  7. Equipment needs for fly husbandry (fly station including a CO2 pad, CO2 supply system, humidity-controlled 25 °C fly incubator)

  8. Spectrophotometer

  9. -80 °C freezer

  10. Rocker for agitation

Procedure

  1. Collecting developmentally synchronized larvae

    1. Cross approximately 200 parental virgin female Drosophila melanogaster and 150 parental males and maintain them on a fly medium sprinkled with instant dry yeast for 2 d at 25 °C.



      Figure 1. Schemes for precise staging. A. Morphological differences between late L2 and early L3 larvae. Comparing to L2 larvae, L3 larvae show 1) larger mouth hooks, 2) branched “finger-like” anterior spiracles, 3) thicker tracheae, and 4) larger orange-color posterior spiracles. In contrast, L2 larvae show 1) smaller mouth hooks, 2) “closed” anterior spiracles, 3) thinner tracheae, and yellow-color posterior spiracles. We mainly rely on the anterior spiracle morphology. B. Scheme of staging. At pre-staging, we collect only L2 larvae on new fly medium plates (pre-staging). Two hours later, we collect third instar larvae in food vials and put the rest of the L2 larvae in a new food plate for the next staging. If required, the larvae can be separated by sex, using either sex-specific genetic markers, such as paternally-inherited X or Y chromosome with a ubiquitous GFP marker that will only be expressed in males (for example see Testa et al., 2013), or using a transgenic sex lethal promoter to drive the expression of GFP in females only, or by assessing the presence or absence of visible gonad (the developing testes, Perry et al., 2014).


    2. To establish the larval cultures, allow the females to lay eggs for 2-4 h on 60 mm tissue culture plates filled with a cornmeal/molasses standard fly medium without any additional yeast at 25 °C. Standardize embryo density to 200 larvae/plate. Excess embryos can be either removed or transferred onto new larval culture plates with a small piece of fly medium to avoid damaging embryos. Due to the high numbers of larvae required for these assays, we recommend establishing a minimum of 4 larval culture plates (approximately 800 larvae) for every time point to be sampled.

    3. Incubate the larval cultures in an incubator at 25 °C and 60% relative humidity under constant light. Constant light conditions are preferred because ecdysone secretion is circadian gated.

    4. Once larvae develop to the end of the second instar (L2; in wildtype, generally 70-72 h after embryo collection), pour 20% sucrose solution (weight/volume) into the larval culture plates. This causes the larvae to float to the surface. We used the morphology of the anterior spiracles to distinguish between second and third instar larvae (Bodenstein, 1950, see also Figure 1A). Remove all third instar larvae and transfer second instar larvae onto a new larval culture plate using either Dumont #3 forceps or entomology forceps. We usually remove all L3 larvae from sucrose solution and transfer L2 larvae by double-checking one by one. This is necessary for pre-staging. (Figure 1B, see also Park et al., 2002, for details of morphological changes during ecdysis in D. melanogaster).

    5. Two hours after transferring second instar larvae, collect all newly-molted L3 larvae into vials and transfer L2 larvae onto another new plate. Raise these newly-molted L3 larvae in a standard plastic fly vial with a normal cornmeal/molasses medium (usually up to 30 larvae/vial) without any additional yeast until the desired developmental time. The time transferred on a new fly medium is set as 0 h after L3 ecdysis (AL3E, see also Figure 1B). If required, newly molted larvae can be separated by sex either by examining the larvae for the presence of visible gonad (the developing testes) or by using genetic markers like paternally-inherited X or Y chromosome that express GFP (only in males, as in Testa et al., 2013) or a transgenic line that uses the sex lethal promoter to drive GFP expression (only in females, Perry et al., 2014).

    6. Repeat this process every two hours until enough larvae have been collected for your experiment. We flood each larval culture plate a maximum of five times, to avoid excess handling of the larvae.


  2. Standard preparation for ecdysone quantification (see also Figure 2)



    Figure 2. A schematic procedure of standard (Procedure B) and sample (Procedure C) preparations


    1. Prepare a 10 µg/µl 20E stock solution in molecular biology or HPLC grade absolute ethanol.

    2. To check the accuracy of the stock solution concentration, dilute the stock solution to 1:200 in absolute ethanol, and measure optical density at 240 nm in a crystal cuvette. The extinction coefficient of 20E in absolute ethanol is 12,656.52 and the molecular weight of 20E is 480.6. Therefore, the formula will be:

      20E concentration (μg/μl) = (OD240) × (Dilution ratio; it will be 200 in this case) × 480.6/12,676.52

    3. Dilute the stock solution to be 1,000 pg/μl, and make a dilution series: 1,000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 pg/μl for the standard curve using absolute ethanol.

    4. Prepare standard solutions by taking 80 µl of the dilution series from Step B3, such that you obtain 80,000, 40,000, 20,000, 10,000, 5,000, 2,500, 1,250, 625 pg/tube respectively. We prepare two replicate tubes for each concentration (see Figure 3).



      Figure 3. Standard curve for ecdysone quantification. Using this curve, we calculated ecdysone concentration as: Ecdysone (pg) = 68,473.88209 × e^(-70.40127 × OD405). R2 = 0.92.


    5. Evaporate ethanol completely using a Speedvac at room temperature simultaneously with Step C9. This typically takes 1-2 h. An additional 10 min may be necessary to completely remove all of the ethanol from the tubes. Pellets are usually invisible.


  3. Sample preparation for ecdysone quantification (see also Figure 2)

    1. At the desired time points, recover staged larvae from the fly medium using a 20% sucrose solution.

    2. Wash collected larvae twice in double-distilled water to remove any fly medium residuals and wash out sucrose solution. Exclude larvae that appear noticeably smaller or larger for their cohort at this step, as it is likely that these larvae have not been correctly staged.

    3. After drying larvae briefly on a small piece of paper towel for about a minute, weigh a group of larvae for one biological replicate together on an ultra-micro balance.

      Usually, 20-30 mg of larvae (50-60 larvae for the onset of the L3 and 35-40 larvae for 24 h after the molt to the L3) is sufficient for accurate ecdysone quantification.

    4. Place all weighed larvae in a 1.5 ml microcentrifuge tube, add three times the volume of absolute methanol (30 μl methanol for 10 mg larvae), and freeze them immediately on dry ice.

    5. Keep all samples at -80 °C until you are ready for ecdysone quantification (Parvy et al., 2005).

    6. When you are ready to begin ecdysone quantification, place all tubes on dry ice and homogenize frozen larvae carefully using disposable pestles and a cordless hand-pestle motor (VWR International).

    7. Centrifuge tubes at 4 °C at maximum speed for 5 min.

    8. Carefully transfer supernatant into new 1.5 ml microcentrifuge tubes. Repeat Steps C7 and C8 1-2 times until there is no visible precipitate.

    9. Evaporate methanol completely using a Speedvac at room temperature for 1-2 h. An additional 10 min of evaporation may be necessary for complete removal of methanol from all tubes. A small precipitate with lipid from the larval fat body should be visible.


  4. Ecdysone quantification by an enzyme immunoassay (EIA)

    1. Redissolve the pellets in 100 µl of EIA buffer. Use only 50 µl of these sample solutions for the assay, saving the remaining 50 µl of this solution. In case the ecdysone concentration of the sample is too high, this sample can be diluted between 2- and 10-fold to be in a range of standard curve.

    2. Set up a plate as outlined in the ecdysone EIA manufacturer’s instructions (Porcheron et al., 1989; https://www.caymanchem.com/pdfs/501390.pdf).

    3. Cover the plate with a piece of parafilm and incubate overnight at 4 °C with gentle agitation.

    4. Reconstitute Ellman’s reagent shortly before use.

    5. Empty the plate by turning it over. Rinse each well with 300 μl wash buffer. Repeat four more times.

    6. After the 5th wash, blot the plate on a few layers of clean paper towel to ensure any liquid will be removed completely.

    7. Add 200 μl of Ellman’s reagent to each 96 well. Make sure the plate is sealed with a piece of parafilm and covered by a piece of aluminum foil, as the Ellman’s reagent is light sensitive. Incubate the plate at room temperature with gentle agitation.

    8. Clean the bottom of the plate with 70% ethanol using paper towels.

    9. Read the plate at a wavelength of 405 nm. Read the plate several times (approximately every 10 min until 60 min have passed post-incubation) to ensure that you capture ideal color development, meaning all standard samples from Step B4 show yellowish color but the color is not yet fully saturated. Multiple readings are necessary because color development continues with time until eventually the color of all samples becomes saturated.

Data analysis

Draw the standard curve as explained in the ecdysone EIA manufacturer’s instructions. Our standard curve for Figure 1A in our publication (Koyama et al., 2014) is shown in Figure 3. Based on this curve, we calculated ecdysone concentration using the following formula:


Ecdysone (pg) = 68,473.88209 × e^(-70.40127 × OD405).


We then corrected the ecdysone concentration in the sample for sample weights obtained in Step C3 to obtain pg ecdysone/mg larval weight.

Notes

Detecting the smaller pulses of ecdysone in third instar larvae requires both accurately staging the larvae from the molt to the third instar and weighing the samples (Warren et al., 2006).

Recipes

  1. Cornmeal/molasses medium (fly medium)/1 L

    1. Weigh and mix all ingredients as below except for Niapagin in a plastic beaker

      Molasses 45 g

      Sugar 75 g

      Cornmeal 70 g

      Yeast Extract 20 g

      Agar 10 g

      Boiling Water 1,100 ml

      10% Niapagin 25 ml

    2. Add boiling water gradually

    3. Transfer the medium into 2 L flasks with screw caps (up to 1,800 ml)

    4. Autoclave at 121 °C for 30 min

    5. When the medium temperature reaches 45-50 °C, add Niapagin

Acknowledgments

We thank for our funding sources, Fundação Calouste Gulbenkian (Portugal) and Australian Research Council (FT170100259) to CKM and the Fundação para a Ciênica e a Tecnologia (Portugal) to TK (SFRH/BPD/74313/2010).

Competing interests

Both TK and CKM do not have any competing interests to declare.

References

  1. Bodenstein, D. (1950). The postembryonic development of Drosophila. In: Biology of Drosophila. Demerec, M. (Ed.). New York: Cold Spring Harbor Laboratory Press, pp. 275-367.
  2. Koyama, T., Rodrigues, M. A., Athanasiadis, A., Shingleton, A. W. and Mirth, C. K. (2014). Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife 3: e03091.
  3. Koyama, T., Texada, M. J., Halberg, K. A. and Rewitz, K. (2020). Metabolism and growth adaptation to environmental conditions in Drosophila. Cell Mol Life Sci 77: 4523-4551.
  4. Park, Y., Filippov, V., Gill, S. S. and Adams, M. E. (2002). Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency. Development 129(2): 493-503.
  5. Parvy, J. P., Blais, C., Bernard, F., Warren, J. T., Petryk, A., Gilbert, L. I., O’Connor, M. B. and Dauphin-Villemantaet, C. (2005). A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster. Dev Biol 282: 84-94.
  6. Perry, J. C., Harrison, P. W. and Mank, J. E. (2014). The ontogeny and evolution of sex-biased gene expression in Drosophila melanogaster. Mol Biol Evol 31: 1206-1219.
  7. Porcheron, P., Moriniere, M., Grassi, J. and Pradelles, P. (1989). Development of an enzyme immunoassay for ecdysteroids using acetylcholinesterase as label. Insect Biochem 19: 117-122.
  8. Testa, N. D., Ghosh, S. M. and Shingleton, A. W. (2013). Sex-specific weight loss mediates sexual size dimorphism in Drosophila melanogaster. PLoS One 8: e58936.
  9. Warren, J. T., Yerushalmi, Y., Shimell, M. J., O'Connor, M. B., Restifo, L. L. and Gilbert, L. I. (2006). Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity. Dev Dyn 235(2): 315-26.

简介

[摘要]类固醇激素严格控制着脊椎动物和无脊椎动物的性成熟时间和最终的体型。在昆虫中,类固醇激素蜕皮激素控制着幼虫龄之间蜕皮的时间以及向蜕变的过渡。最终龄期间生长占昆虫增加最终质量的80%以上,而这种生长周期的持续时间是由小的序列驱动的蜕皮激素脉冲,最终诱导metamo ř phosis。从历史上讲,蜕皮激素的生物活性形式20-hydroxyec dysone(20E)是使用放射免疫分析,生物分析或色谱分析定量的。但是,这些测定方法学上很复杂并且通常很耗时。此外,在像果蝇(Drosophila melanogaster)这样的小昆虫中,使用这些测定法收集样品以准确测定蜕皮酮的浓度是有限的。在这里,我们描述了一种精确而灵敏的方法,用于收集精心准备的三龄幼虫,这些幼虫适用于准备使用商用20E酶免疫法(EIA)进行蜕皮激素定量的样品。因为我们使幼虫蜕皮与最终龄期重新同步,收集大量样品并称重每个样品,所以我们能够在最终龄期早期检测到一个小的蜕皮激素峰,称为临界重量蜕皮激素峰。该方法可检测低至6 pg 20E / mg幼虫样品的峰,使我们能够定量测定果蝇中的其他蜕皮激素小峰,这是最终确定其调控和功能的必要前提。

[背景]在BRO一个的d范围动物,发展是紧密由类固醇激素调节。在哺乳动物中,青春期的发作主要受性类固醇激素的调节(Koyama等,2020)。在昆虫中,类固醇激素的蜕皮激素调节的moltin定时克,inclu丁所述的定时变质蜕皮。由于浓度波动小蜕皮激素预计将触发键发育事件,发展的方法来检测改变我ñ蜕皮激素浓度是科学家,旨在评估类固醇激素的作用是至关重要的。在该协议中,我们描述了如何从发育同步的幼虫中收集高质量的样品,以及如何使用市售的酶免疫分析试剂盒为蜕皮激素定量制备这些样品。

关键字:20-羟基蜕皮酮, 幼虫同步, 幼虫分期, 酶免疫分析, 黑腹果蝇, 全身脱皮激素定量, 临界体重

材料和试剂

1.5 ml微量离心管

纸巾
Parafilm (Bemis Company,PM996)
铝箔
一次性杵
水晶比色杯
200个父母初生女性果蝇(Drosophila melanogaster)和150个父母男性
预涂(鼠抗兔IgG抗体)ELISA 96孔条板(Cayman Chemicals公司,目录号:400005 ),小号撕4 ℃下
购买后长达一年,我们已经成功使用了这些印版。

绝对甲醇(VWR,甲醇绝对≥99.8%ACS,目录号:M1240-4LTGL),在室温下储存
绝对乙醇(VWR,乙醇绝对值≥99.8%,Electran分子生物学等级,目录号:437435L),在室温下存储
20-羟基蜕皮激素EIA试剂盒(Cayman Chemicals,目录号:501390)
20-羟基蜕皮酮(SciTech Chemicals,目录号:S3314-001)
蔗糖(50克蔗糖在250毫升ddH 2 O中的蔗糖。最好是新鲜制备的。否则应在4 °C下保存长达一周。使用前应加热到约20-25°C )
饲养管理所需的材料(干酵母,一次性塑料瓶,塞子,60 mm组织培养板,胚胎收集室)
双蒸馏水
干冰
70%乙醇
糖蜜

棒子面
酵母抽提物
琼脂
玉米面/糖蜜培养基(果蝇培养基,请参见食谱)

设备


Dumont#3 Force ps(精细科学工具,目录号:11231-30)或昆虫学钳(Fisher Scientific,目录号:S72110)
超微量天平(Sartorius,Sartorius Cubis系列,SE2)
无线杵式电动机(VWR International,VWR无线杵式电动机,目录号:47747-370)
冷冻微量离心机
SpeedVac (Savant,SpeedVac Plus SC10A,目录号:SC110A-8E210146-4K )
酶标仪,Victor3(PerkinElmer ,Victor3,目录号:1420-050 )
畜牧业的设备需求(包括CO 2垫,CO 2供给系统,湿度控制的25°C苍蝇孵化器的苍蝇站)
分光光度计
-80°C冷冻室
摇杆摇动

程序
收集发育同步的幼虫
将大约200个亲本原始雌性果蝇(Drosophila melanogaster)和150个亲本杂交,并在撒有速溶干酵母的果蝇培养基上保持25℃2天。

图1.精确分级的方案。一。L2晚期和L3早期幼虫之间的形态学差异。与L2幼虫相比,L3幼虫显示1)较大的钩形钩,2)分支成“手指状”的前气管,3)较粗的气管,和4)较大的橙色后气管。相反,L2幼虫显示1)较小的嘴钩,2)“闭合”的前呼吸道,3)气管较薄,后呼吸道呈黄色。我们主要依靠前鼻梁形态。乙。分期方案。在预分期中,我们仅在新的蝇蝇培养基板上收集L2幼虫(预分期)。两个小时后,我们收集在食品小瓶第三龄幼虫,并把该二层的休息啦rvae新型食品板的下一次升级。如果需要,可以使用性别特异性的遗传标记将幼虫按性别分开,例如具有普遍存在的GFP标记的父本遗传的X或Y染色体,仅在男性中表达(例如,参见Testa et al 。,2013)。 ),或使用转基因性致死启动子仅在或通过评估可见性腺的存在或不存在,以驱动GFP的女性表达式(显影睾丸,佩里等人。,2014)。


要建立幼虫培养物,让雌性在25°C的情况下在装有玉米粉/糖蜜标准果蝇培养基的60 mm组织培养板上产卵2-4小时,而无需添加任何酵母。Standardi ž ë胚胎密度到200幼虫/板。多余的胚胎可以移出或转移到带有一小段飞行培养基的新的幼虫培养板上,以免损坏胚胎。由于这些测定需要大量幼虫,因此我们建议在每个采样时间至少建立4个幼虫培养板(约800个幼虫)。
在恒定光照下于25°C和60%相对湿度的培养箱中孵育幼虫培养物。优选恒定的光照条件,因为蜕皮激素的分泌是昼夜节律的。
一旦幼虫发育到第二龄期的末尾(L2;在野生型中,通常在收集胚胎后70-72小时),将20%的蔗糖溶液(重量/体积)倒入幼虫培养板中。这会使幼虫漂浮到水面。我们使用前呼吸道的形态来区分第二龄和第三龄幼虫(Bodenstein ,1950,另请参见图1A )。取出所有三龄幼虫,然后使用Dumont#3镊子或昆虫学镊子将第二龄幼虫转移到新的幼虫培养板上。通常,我们从蔗糖溶液中取出所有L3幼虫,并通过一一仔细检查来转移L2幼虫。这对于预升级是必需的。(图1B,也参见公园等人,2002,为的形态变化细节在蜕皮在黑腹果蝇)。
转移第二龄幼虫两小时后,将所有新蜕变的L3幼虫收集到小瓶中,然后将L2幼虫转移到另一个新板上。在标准的塑料蝇管小瓶中用普通的玉米粉/糖蜜培养基(通常最多30个幼虫/小瓶)饲养这些新近蜕皮的L3幼虫,直至达到所需的发育时间。个新飞介质上转印E时间被设定为L3后0 H蜕皮(AL3E,也参见图1B)。如果需要,可以通过检查幼虫是否存在可见的性腺(发育中的睾丸)或使用遗传标记(如父本遗传的表达GFP的X或Y染色体)(仅在雄性中,如雄性)按性别将新蜕变的幼虫按性别分开。种皮等人。,2013)或转基因品系,它使用性致死启动子来驱动的GFP表达(仅在雌性,佩里等人。,2014)。
每两个小时重复一次此过程,直到为实验收集到足够的幼虫为止。我们将每个幼虫培养板最多浸入5次,以避免过多处理幼虫。

蜕皮激素定量的标准制备方法(另见图2)

图2.标准(过程B)和样品(过程C)制备的示意性过程

准备分子生物学或HPLC级无水乙醇的10 µg / µl 20E储备溶液。
要检查原液浓度的准确性,请在无水乙醇中将原液稀释至1:200,并在比色皿中测量240 nm处的光密度。20E的在无水乙醇的消光系数为12 ,656.52和所述20E的分子量为480.6。因此,公式将是:
20E浓度(μ克/ μ升)=(OD 240 )×(稀释比例;它将是200在这种情况下)× 480.6 / 12 ,676.52

稀释原液为1 ,000微克/ μ升,并进行稀释系列:1 ,000,500,250,125,62.5,31.25,15.625,和7.8125微克/ μ升用于使用绝对标准曲线乙醇。
通过取80制备标准溶液微升的稀释系列从步骤B3,使得你获得80000,40000,20000,10000 5000,2500,1 ,250,625微克/分别管。对于每种浓度,我们准备两个重复试管(见图3)。

图3.蜕皮激素定量的Stan dard曲线。使用该曲线,我们计算蜕皮激素浓度为:蜕皮激素(PG )= 68 ,473.88209 × È ^(-70.40127 × OD 405 )。R 2 = 0.92。

在室温下与步骤C9同时使用Speedvac完全蒸发乙醇。这通常需要1-2小时。要从试管中完全除去所有乙醇,可能还需要10分钟。药丸通常是看不见的。

样品制备FO ř蜕皮激素定量(也参见图2)
在期望的时间点,恢复从使用蝇介质上演幼虫一个20%的蔗糖溶液。
用双蒸馏水将收集的幼虫洗两次,以除去任何苍蝇培养基残留物,并洗净蔗糖溶液。在此步骤中,排除因其同类群而显得明显较小或较大的幼虫,因为这些幼虫可能未正确分阶段。
在一小块纸巾上短暂干燥幼虫约一分钟后,称重一组幼虫,以超微量天平一起进行一次生物复制。
通常,幼虫20-30 mg(L3发作时为50-60幼虫,L3蜕变后为24h时为35-40幼虫)足以对蜕皮激素进行准确定量。

将所有称重幼虫在1.5ml微量离心管中,加第REE倍无水甲醇(30体积μ升甲醇,10毫克幼虫),并立即将它们冷冻在干冰上。
将所有样品保持在-80 °C,直到您准备进行蜕皮激素定量(Parvy等,2005)。
准备开始进行蜕皮激素定量时,将所有试管放在干冰上,并使用一次性杵和无绳手摇马达(VWR International)小心地将冷冻幼虫匀浆。
              将离心管在4 °C下以最大速度离心5分钟。
              小心地将上清液转移到新的1.5 ml微量离心管中。重复步骤s C7和C 8 1-2次,直到没有可见的沉淀为止。
              在室温下使用Speedvac将甲醇完全蒸发1-2小时。为了从所有管中完全除去甲醇,可能还需要再蒸发10分钟。应该可以看到幼虫脂肪体内有少量的脂质沉淀。
通过酶免疫法(EIA)对蜕皮激素进行定量
              再溶解丸粒在100μl的EIA缓冲液中。仅将这些样品溶液中的50 µl用于分析,节省剩下的50 µl该溶液。如果样品中的蜕皮激素浓度过高,可以将该样品稀释2倍至10倍,以使其在标准曲线范围内。
按照蜕皮激素EIA制造商的说明(Porcheron et al。,1989; https://www.caymanchem.com/pdfs/501390.pdf)概述设置板。
用一块封口膜覆盖板,并在4 °C下温和搅拌孵育过夜。
在使用前不久重新配制Ellman的试剂。
翻转将盘子倒空。冲洗用300各孔微升洗涤缓冲液。再重复四遍。
第5次清洗后,将板用几层干净的纸巾吸干,以确保将所有液体彻底清除。
加入200微升的埃尔曼的试剂每个96孔。由于Ellman的试剂对光敏感,因此请确保用一块封口膜密封板并用一块铝箔纸覆盖。在室温下轻轻摇动孵育板。
用纸巾用70%乙醇清洁板的底部。
读板在405 nm的波长。读板数次(约每10分钟至60分钟已通过后-孵化),以确保您捕捉理想的发色,这意味着从B4步骤所有的标准样本显示颜色偏黄,但颜色还没有完全饱和。必须进行多次读数,因为颜色会随着时间持续发展,直到最终所有样品的颜色饱和为止。

数据分析


按照蜕皮激素EIA制造商的说明中的说明绘制标准曲线。我们对图1A中的出版物标准曲线(小山。等人,2014)被示出在基于该曲线上的图3,我们计算蜕皮激素使用下列公式浓度:


蜕皮激素(PG )= 68 ,473.88209 × E 1( - 70.40127 × OD 405 )。


然后,针对在步骤C3中获得的样品重量,校正样品中的蜕皮激素浓度,以获得pg蜕皮激素/ mg幼虫重量。


笔记


要检测第三龄幼虫中蜕皮激素的较小脉冲,既需要准确地将幼虫从蜕皮转移到第三龄幼虫,也需要对样品进行称重(Warren等,2006)。


菜谱


1.玉米面/糖蜜培养基(果蝇培养基)/ 1 L     
一种。称量并混合以下所有成分(将Niapagin除外)放入塑料烧杯中     
糖蜜45克

糖75克

玉米面70克

酵母提取物20克

琼脂10克

沸水1 ,百米升

10%尼apagin 25百万升

b。逐渐加入沸水     
C。介质转移到2个与螺旋盖升锥形瓶中(高达1 ,800米升)     
d。在121 °C下高压灭菌30分钟     
e。当介质温度达到45-50 °C时,添加Niapagin     

致谢


我们感谢我们的资金来源,Fundação卡洛斯提古尔本基安(葡萄牙)和澳大利亚研究理事会(FT170100259),以CKM和Fundação第一个Ciênica EA TECNOLOGIA (葡萄牙)以TK(SFRH / BPD /二千○十分之七万四千三百十三)。


利益争夺


TK和CKM都没有任何竞争利益要声明。


参考文献


Bodenstein,D.(1950年)。果蝇的胚胎后发育。在:果蝇生物学。Demerec ,M。(编。)。纽约:冷泉港实验室出版社,第275 - 367。
Koyama,T.,Rodrigues,MA,Athanasiadis,A.,Shingleton,AW和Mirth,CK(2014)。通过FoxO-Ultraspiracle介导的蜕皮激素生物合成来控制体重的营养。Elife 3 :e03091。
Koyama,T.,Texada,MJ,Kalberg,KA和Rewitz,K.(2020)。果蝇的代谢和生长适应环境的影响。Cell Mol Life Sci 77 :4523-4551。
Park,Y.,Filippov,V.,Gill,SS和ME ,Adams (2002 )。蜕皮触发激素基因的缺失导致致命的蜕皮缺乏。发展129 (2):493 - 503。
Parvy,JP,Blais,C.,Bernard,F.,Warren,JT,Petryk,A.,Gilbert,LI,O'Connor,MB和Dauphin-Villemantaet,C。(2005)。βFTZ-F1在黑腹果蝇胚胎后发育过程中调节蜕皮类固醇滴度中的作用。Dev Biol 282 :84-94。
佩里(J.C.)佩里(Jerry),哈里森(P.)哈里森(Harrison)和JE(英国)曼克(Mank,JE)(2014)。黑腹果蝇性别偏向基因表达的发生与发展。Mol Biol Evol 31:1206-1219。
Porcheron,P.,Moriniere,M.,Grassi,J。和Pradles,P。(1989)。以乙酰胆碱酯酶为标记的蜕皮类固醇酶免疫测定方法的开发。昆虫生物化学19 :117-122。
Testa,ND,Ghosh,SM和Shingleton,AW(2013)。特定性别的体重减轻介导果蝇的性大小二态性。PLoS One 8:e58936。
Warren,JT,Yerushalmi,Y.,Shimell,MJ,O'Connor,MB,Restifo,LL和Gilbert,LI(2006年)。果蝇后期幼虫发育过程中蜕皮激素20-羟基蜕皮酮的离散脉冲:与基因活性的变化相关。Dev Dyn 235 (2):315-26。
  • 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 Koyama and Mirth. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Koyama, T. and Mirth, C. K. (2021). Ecdysone Quantification from Whole Body Samples of Drosophila melanogaster Larvae. Bio-protocol 11(3): e3915. DOI: 10.21769/BioProtoc.3915.
  2. Koyama, T., Rodrigues, M. A., Athanasiadis, A., Shingleton, A. W. and Mirth, C. K. (2014). Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife 3: e03091.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。