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Jan 2018
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Detection of Cell Death in Planarians
真涡虫细胞死亡的检测   

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Abstract

Planarians are freshwater flatworms, well known for their ability to regenerate a complete organism from any piece of their body. Furthermore, planarians are constantly growing and degrowing throughout their lives, maintaining a functional and proportioned body. These properties rely on the presence of a population of adult stem cells and on the tight control of their cell renewal, which is based on the balance between the proliferation of new cells and their differentiation, and the death of unnecessary cells. Due to the importance of these two processes in planarian biology, over the years, researchers have optimized molecular techniques to detect both cell proliferation and cell death in planarians. Here, we present the two main protocols currently used for cell death detection and quantification in the planarian field: Caspase-3 activity quantification and TUNEL assay.

Keywords: Caspase-3 activity (Caspase-3活性), TUNEL assay (TUNEL分析), Planarians (真涡虫), Cell death (细胞死亡), Cell turnover (细胞更新)

Background

Cell renewal in adult organisms is a complex mechanism based on three processes: (a) the elimination of selected cells by cell death; (b) the replacement of eliminated cells through cell division, typically involving adult stem cells and their descendants; and (c) the differentiation of newly generated cells and their integration with preexisting tissue (Pellettieri and Sanchez Alvarado, 2007; González-Estévez and Saló, 2010). In planarians, cell renewal must be continuously coordinated, since they grow and degrow depending on food availability and temperature (Baguñá and Romero, 1981). It is known that the changes in size result mainly from changes in cell number, rather than in cell size, so the ratio of dying/proliferating cells is controlled by environmental conditions (González-Estévez and Saló, 2010). Planarians are able to tolerate long starvation periods, and during this time, they degrow up to minimum sizes. Under these stressful conditions, food reserves from gastrodermis and mesenchyma are the firsts to be used, and at more extreme points, the sexual strains digest the sexual organs, and become asexual (González-Estévez and Saló, 2010; Miller and Newmark, 2012). When food is available, planarians are able to grow back, and in the sexual strains, the reproductive organs reappear. These cycles of grow and degrow occur throughout planarian lives without damage to the animal.

During planarian starvation, cell death increases to re-organize the organs and structures, and planarian adult stem cells (neoblasts) self-renewal is maintained at basal levels, resulting in a decrease of planarian body size (Figures 1A and 1B) (González-Estévez et al., 2012). The tissue remodeling is critical during planarian starvation because it maintains a proportioned planarian body. It was shown that JNK signaling, and Gtdap-1 are controlling the planarian body re-scaling during degrowing through the modulation of apoptotic cell death (González-Estévez et al., 2007; Almuedo-Castillo et al., 2014).

Because cell death is a relevant process in planarian, in the last few years molecular techniques to detect and measure cell death have been developed and optimized. Here, we will explain step-by-step the two main protocols used to detect cell death in planarians: measurement of Caspase-3 activity and TUNEL assay.


Figure 1. Planarian homeostasis. A. Planarians are able to grow and degrow during their lives, maintaining their body proportions and functionality. Image provided by Gustavo Rodriguez-Esteban. B. After a stimulus, proliferation and/or cell death can change in planarians. After feeding, neoblast proliferation increases throughout planarian body, and cell death is reduced to minimum levels, resulting in the increase of animal’s size. Conversely, when planarians are in starvation, neoblast proliferation is maintained at basal levels and cell death increases, which not only results in a decrease in body size but allows the reorganization of the tissues. Image from Nídia de Sousa Ph.D. thesis (de Sousa, 2017).


Part I: Caspase-3 activity assay

The Caspase-3 activity assay is a fluorescent assay that detects the activity of Caspase-3 in cell lysates using the fluorogenic substrate acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC). It is based on the hydrolysis of Ac-DEVD-AMC by Caspase-3, resulting in the release of the fluorescent 7-amino-4-methylcoumarin (AMC). AMC that can be detected using a luminescence spectrophotometer with excitation at 380 nm and emission between 420 nm and 460 nm. Cleavage of the substrate only occurs in lysates in which Caspase-3 is present, which is a gene required for apoptosis; therefore, the amount of AMC produced is proportional to the number of apoptotic cells in the sample.

Materials and Reagents

  1. Petri dish (VWR, catalog number: 391-0439 )
  2. Slides (VWR, catalog number: 631-1551 )
  3. Razor blade (MARTOR, catalog number: NO. 743 )
  4. Eppendorf tubes (VWR, Eppendorf, catalog number: 700-5239
  5. 15 ml Falcons (LF Equipamentos, catalog number: 166 )
  6. Spectrophotometry Cuvettes (VWR, catalog number: 634-0677BTU )
  7. 96-well plate (VWR, Corning, catalog number: 734-1664 )
  8. MilliQ water
  9. Ice
  10. Micro BCA Protein Assay Kit (Thermo Fisher Scientific, PierceTM, catalog number: 23235 )
  11. Tris-HCl, pH 8 (Sigma-Aldrich, catalog number: 93362 )
  12. EDTA, pH 8 (Sigma-Aldrich, catalog number: 1233508 )
  13. Triton X-100 (Sigma-Aldrich, catalog number: X100
  14. HEPES pH 7.5 (Sigma-Aldrich, catalog number: H3375
  15. Glycerol 10% (Sigma-Aldrich, catalog number: G5516 )
  16. DTT (Sigma-Aldrich, catalog number: 646563 )
  17. Caspase-3 inhibitor Z-DEVD-FMK (Merck, catalog number: 264155 )
  18. Caspase-3 substrate Ac-DEVD-AMC (BD Biosciences, PharmingenTM, catalog number: 556449 )
  19. Lysis buffer (see Recipes)
  20. Assay buffer (see Recipes)

Equipment

  1. Oven
  2. Luminescence spectrophotometer
  3. Pipettes
  4. Vortex
  5. Centrifuge
  6. Platform shaker
  7. 4 °C refrigerator
  8. -20 °C freezer
  9. 37 °C incubator

Procedure

  1. Protein extraction (Figure 2A)
    1. Prepare lysis buffer (see Recipe 1)
    2. In a Petri dish with ice, place a slide and transfer planarians to the slide. Remove the excess of water and chop planarians into small pieces with a razor blade (removing the mucus from the razor blade with a piece of paper when necessary).
      Note: A minimum of 5 planarians with 3-5 mm per condition.
    3. Add 100 μl of lysis buffer per planarian to the slide and use it to aspirate planarian fragments into an Eppendorf tube. Keep it on ice. 
    4. Homogenize by pipetting with a P200 and vortexing for 5 sec, maintaining, whenever possible, the Eppendorf tubes on ice.
    5. Centrifuge at 13,000 x g for 10 min at 4 °C.
    6. Transfer the supernatant fraction to a new tube at 4 °C. For long-term storage keep at -20 °C.

  2. Protein concentration determination using Micro BCA Protein Assay Kit (Figure 2B)
    1. Perform the necessary BSA protein dilutions to obtain the standard curve following the manufacturer’s recommendations.
    2. Prepare the working reagent following the manufacturer’s recommendations.
    3. Dilute your samples 250 times (e.g., 2 μl of sample + 498 μl MilliQ water).
    4. To each spectrophotometry cuvette, add 500 μl of Working Reagent + 500 μl of diluted sample. This procedure should also be done to the samples used to obtain the standard curve.
    5. Incubate the spectrophotometry cuvettes in an oven for 1 h at 60 °C. 
    6. Remove the samples from the oven and measure the absorbance (λ = 562 nm) in a spectrophotometer.
      Note: All the samples should be read in no more than 10 min.
    7. Construct a standard curve using the values of the protein dilutions performed in Step B1. Subtract the value of the blank sample (with no protein, obtained in Step B1) to avoid background measures. Calculate the protein concentration of your samples. Multiply the value obtained for the dilution factor.

  3. Caspase-3 activity measurement (Figure 2C)
    1. Prepare the assay buffer (see Recipe 2)
    2. Load the 96-well plate. 
    3. Start with the blank wells. The blank solution is composed of 125 μl assay buffer + 25 μl of lysis buffer. Blank wells must be done in triplicate.
      Note: All the samples loaded in 96-well plate must be done in triplicate. This means that for each condition (blank, negative control and samples) you must have at least three wells.
    4. Load the experimental negative control. For the negative control, protein from a control sample should be used. Load in each well 123 μl of assay buffer + 20 μg of protein (x μl of protein extract + y μl of lysis buffer until 25 μl of volume) + 1.5 μl of Caspase-3 inhibitor Z-DEVD-FMK. Negative controls must be done in triplicate.
      Note: Experimental control is used to confirm that you are measuring Caspase-3 activity. Using the Caspase-3 inhibitor is supposed to obtain low levels of Caspase-3 activity. It is essential to have one control but as many as desired can be included.
    5. In the remaining wells load 123 μl of assay buffer + 20 μg of protein (x μl of protein extract + y μl of lysis buffer to 25 μl of final volume). All the samples must be done in triplicate.
    6. Incubate the plate for 15 min in the dark at 37 °C, in agitation.
    7. Load 2 μl of Caspase-3 substrate Ac-DEVD-AMC to all plate wells and incubate for 2 h in the dark at 37 °C.
    8. Measure enzyme activity through luminescence, using a luminescence spectrophotometer (excitation 380 nm, emission 440 nm).


    Figure 2. General view of Caspase-3 assay protocol. A. Cartoon exemplifying protein extraction. B. Step-by-Step of protein concentration determination. C. Example of how load plate wells.

Data analysis

  1. Average the three technical replicates to obtain a single read to each sample.
  2. Subtract the value of the blank sample to avoid background reads.
  3. Plot the negative controls, experimental controls, and target samples.

Part II: TUNEL assay

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that present extensive DNA degradation. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template.

Materials and Reagents

  1. Glass vials (Sigma-Aldrich, catalog number: 27151 )
  2. 96-well plate (VWR, catalog number: 734-2781 )
  3. Glass slide (VWR, catalog number: 631-1551 )
  4. Coverslip (Merck, catalog number: S7117 )
  5. ApopTag Red In Situ Apoptosis Detection Kit (Merck, catalog number: S7165 )
  6. 10% n-acetyl cysteine (diluted in PBS 1x) (Sigma-Aldrich, catalog number: A9165 )
  7. PBS 1x
  8. 37% formaldehyde (Merck, catalog number: 104003 )
  9. 1% SDS (diluted in PBS 1x) (Sigma-Aldrich, catalog number: L3771 )
  10. 30% H2O2 (Merck, catalog number: 107209 )
  11. Bleaching solution
  12. TdT enzyme (from ApopTag Kit)
  13. Anti-digoxigenin-rhodamine (from ApopTag Kit)
  14. 70% glycerol (diluted in PBS 1x)
  15. PBSTx (see Recipes)
  16. PBSTB (see Recipes)

Equipment

  1. Platform shaker
  2. Pipettes
  3. 37 °C incubator
  4. 4 °C refrigerator
  5. Confocal microscope

Software

  1. ImageJ

Procedure

  1. Place animals in glass vials and remove the planarian water using a pipette.
    Note: Animals should be between 3 mm and 5 mm large.
  2. Incubate the animals with 5 ml of 10% N-acetyl cysteine (diluted in PBS 1x) for 5 min at RT, rocking.
    Notes: 
    1. Although glass vials are rocking, ensure that planarians are properly separated and in contact with the solution.
    2. This step kills the animals and removes their thick coating of mucus.
  3. Remove N-acetyl cysteine and fix the animals with 5 ml of 4% formaldehyde (diluted in PBSTx) for 20 min at RT, rocking.
    Note: Every liquid exchange should be performed carefully through pipetting but not touching planarians with the tips, to avoid tissue damage.
  4. Remove the fixative and incubate the animals with 5 ml of 1% SDS (diluted in PBS 1x) for 20 min at RT, rocking.
  5. Remove the previous solution and wash animals twice with 5 ml of PBSTx for 5 min each at RT, rocking.
  6. Remove the washing solution and bleach animals with 5 ml of 6% H2O2 (diluted in PBSTx), overnight under white light, without rocking.
    Note: An overnight is around 16 h. Please be sure to perform this bleaching time.
  7. Remove the bleaching solution and wash the animals twice with 5 ml of PBSTx for 5 min each at RT, rocking.
    Note: On this step, animals can be stored at 4 °C for a few days (no longer than 3 days).
  8. Remove the washing solution and rinse animals in 5 ml of PBS 1x.
  9. Carefully, transfer the animals to the 96-well plate.
    Note: It is important to be sure that all animals in the well are in contact with the solution. If you have several animals, it is better to distribute them for two or three wells.
  10. Using a pipette remove PBS 1x and incubate the animals with 100 μl of TdT enzyme diluted in Reaction buffer (both from the ApopTag Kit) for 4 h at 37 °C, rocking.
  11. Remove the TdT enzyme and rinse animals with 100 μl of stop/wash buffer (from the ApopTag Kit). 
  12. Remove the previous solution and wash animals twice with 100 μl of PBSTB (PBSTx with 0.25% BSA) for 5 min at RT, rocking.
  13. Remove the washing solution and stain animals with 100 μl of anti-digoxigenin-rhodamine diluted in Blocking solution (both from the ApopTag Kit) for 4 h at RT, on dark, rocking.
    Note: From this step until the end of the protocol, animals should be maintained in the dark.
  14. Remove the previous solution and wash stained animals 4 times with 100 μl of PBSTB each for 10 min at RT, rocking. Keep washing overnight in PBSTB at 4 °C, rocking.
  15. Wash animals until obtain a clear signal (Figure 3) with PBSTB at RT, rocking.
    Note: Change the solution every 20/30 min.
  16. Remove the previous solution and add 70% glycerol to the animals.
  17. Transfer the animals to a glass slide and mount with a coverslip. Add glycerol if necessary.
    Note: To avoid glycerol evaporation and sample disruption, apply transparent nail varnish around the coverslip. Be aware to not apply varnish over the animals.


    Figure 3. Result of a TUNEL assay. The image shows the tail of a planarian imaged by confocal microscopy. White dots correspond to nucleus of dying cells. Scale bar = 250 μm.

Data analysis

  1. Acquire images using a confocal microscope. 
  2. Analyse the images in ImageJ software.

Recipes

  1. Lysis buffer
    Tris-HCl (pH 8) 5 mM
    EDTA (pH 8) 20 mM
    Triton 0.5%
    Reserve the lysis buffer on ice until it is necessary
  2. Assay buffer
    HEPES (pH 7.5) 20 mM
    Glycerol 10%
    DTT 2 mM
    Reserve the assay buffer at RT until it is necessary
  3. PBSTx
    1x PBS with 0.3% Triton
  4. PBSTB
    PBSTx with 0.25% BSA

Acknowledgments

This work was supported by grant BFU2008-01544 and BFU2014-56055-P (Ministerio de Educación y Ciencia) and grant 2009SGR1018 (AGAUR). N.S. was supported by the APIF fellowship from the Universitat de Barcelona. Caspase-3 activity measurement and TUNEL assay protocol were adapted from the González-Estévez et al. (2007) and Pellettieri et al. (2010), respectively.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Almuedo-Castillo, M., Crespo-Yanez, X., Seebeck, F., Bartscherer, K., Salo, E. and Adell, T. (2014). JNK controls the onset of mitosis in planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling. PLoS Genet 10(6): e1004400.
  2. Baguñá, J. and Romero, R. (1981). Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84: 181-194.
  3. de Sousa, N. (2017). Ph.D. Thesis. Role of Hippo pathway in planarians. University of Barcelona.
  4. González-Estévez, C. and Salo, E. (2010). Autophagy and apoptosis in planarians. Apoptosis 15(3): 279-292.
  5. González-Estévez, C., Felix, D. A., Aboobaker, A. A. and Salo, E. (2007). Gtdap-1 promotes autophagy and is required for planarian remodeling during regeneration and starvation. Proc Natl Acad Sci U S A 104(33): 13373-13378.
  6. González-Estévez, C., Felix, D. A., Rodriguez-Esteban, G. and Aboobaker, A. A. (2012). Decreased neoblast progeny and increased cell death during starvation-induced planarian degrowth. Int J Dev Biol 56(1-3): 83-91.
  7. Miller, C. M. and Newmark, P. A. (2012). An insulin-like peptide regulates size and adult stem cells in planarians. Int J Dev Biol 56(1-3): 75-82.
  8. Pellettieri, J. and Sanchez Alvarado, A. (2007). Cell turnover and adult tissue homeostasis: from humans to planarians. Annu Rev Genet 41: 83-105.
  9. Pellettieri, J., Fitzgerald, P., Watanabe, S., Mancuso, J., Green, D. R. and Sanchez Alvarado, A. (2010). Cell death and tissue remodeling in planarian regeneration. Dev Biol 338(1): 76-85.

简介

涡虫是淡水扁虫,因其能够从身体的任何部分再生完整的有机体而闻名。 此外,涡虫在其一生中不断生长和去除,保持功能和比例的身体。 这些特性依赖于成体干细胞群的存在以及对细胞更新的严格控制,其基于新细胞增殖与其分化之间的平衡以及不必要细胞的死亡。 由于这两个过程在涡虫生物学中的重要性,多年来,研究人员已经优化了分子技术,以检测涡虫中的细胞增殖和细胞死亡。 在这里,我们提出了目前用于细胞死亡检测和量化的两种主要方案:Caspase-3活性定量和TUNEL分析。
【背景】成体生物体中的细胞更新是基于三个过程的复杂机制:(a)通过细胞死亡消除选定的细胞; (b)通过细胞分裂取代已消除的细胞,通常涉及成体干细胞及其后代; (c)新生细胞的分化及其与先前存在的组织的整合(Pellettieri和Sanchez Alvarado,2007;González-Estévez和Saló,2010)。在涡虫中,细胞更新必须不断协调,因为它们根据食物的可用性和温度而生长和蜕皮(Baguñá和Romero,1981)。众所周知,大小的变化主要是由于细胞数量的变化而不是细胞大小的变化,因此死亡/增殖细胞的比例受环境条件的控制(González-Estévez和Saló,2010)。涡虫能够忍受长期饥饿期,并且在此期间,它们可以延长至最小尺寸。在这些压力条件下,来自胃肠道和间充质的食物储备是第一次使用,在更极端的地方,性毒株消化性器官,变成无性(González-Estévez和Saló,2010; Miller和Newmark,2012) 。当食物可用时,涡虫能够生长,并且在性变种中,生殖器官重新出现。这些生长和蜕皮周期发生在整个涡虫生命中,而不会对动物造成伤害。

在涡虫饥饿期间,细胞死亡增加以重新组织器官和结构,并且涡虫成体干细胞(新生细胞)自我更新维持在基础水平,导致涡虫体尺寸减小(图1A和1B)(González- Estévez et al。,2012)。在涡虫饥饿期间,组织重塑是至关重要的,因为它保持了比例的涡虫体。结果表明,JNK信号传导和 Gtdap-1 通过调节凋亡细胞死亡来控制胚胎体重新扩大(González-Estévez等。, 2007; Almuedo-Castillo et al。,2014)。

由于细胞死亡是涡虫中的相关过程,因此在过去几年中已经开发并优化了用于检测和测量细胞死亡的分子技术。在这里,我们将逐步解释用于检测涡虫中细胞死亡的两种主要方案:Caspase-3活性的测量和TUNEL测定。


图1. Planarian稳态。 A. Planarians能够在生命中成长和蜕皮,保持身体比例和功能。图片由Gustavo Rodriguez-Esteban提供。 B.刺激后,增殖和/或细胞死亡可能会在涡虫中发生变化。喂食后,整个涡虫体内成纤维细胞增殖增加,细胞死亡率降至最低水平,导致动物体积增大。相反,当涡虫处于饥饿状态时,成纤维细胞增殖维持在基础水平并且细胞死亡增加,这不仅导致体尺减小而且允许组织的重组。来自NídiadeSousa博士的图片论文(de Sousa,2017)。

关键字:Caspase-3活性, TUNEL分析, 真涡虫, 细胞死亡, 细胞更新


第一部分:Caspase-3活性检测

Caspase-3活性测定是荧光测定,其使用荧光底物乙酰基Asp-Glu-Val-Asp 7-酰氨基-4-甲基香豆素(Ac-DEVD-AMC)检测细胞裂解物中Caspase-3的活性。它基于Caspase-3对Ac-DEVD-AMC的水解,导致荧光7-氨基-4-甲基香豆素(AMC)的释放。可以使用发光分光光度计检测AMC,其中激发波长为380nm,发射波长为420nm至460nm。底物的裂解仅发生在存在Caspase-3的裂解物中,Caspase-3是凋亡所需的基因;因此,产生的AMC的量与样品中凋亡细胞的数量成比例。

材料和试剂

  1. 培养皿(VWR,目录号:391-0439)
  2. 幻灯片(VWR,目录号:631-1551)
  3. 剃刀刀片(MARTOR,目录号:NO.743)
  4. Eppendorf管(VWR,Eppendorf,目录号:700-5239) 
  5. 15毫升猎鹰(LF Equipamentos,目录号:166)
  6. 分光光度法比色皿(VWR,目录号:634-0677BTU)
  7. 96孔板(VWR,Corning,目录号:734-1664)
  8. MilliQ水
  9. Micro BCA蛋白质分析试剂盒(Thermo Fisher Scientific,Pierce TM ,目录号:23235)
  10. Tris-HCl,pH 8(Sigma-Aldrich,目录号:93362)
  11. EDTA,pH 8(Sigma-Aldrich,目录号:1233508)
  12. Triton X-100(Sigma-Aldrich,目录号:X100) 
  13. HEPES pH 7.5(Sigma-Aldrich,目录号:H3375) 
  14. 甘油10%(西格玛奥德里奇,目录号:G5516)
  15. DTT(Sigma-Aldrich,目录号:646563)
  16. Caspase-3抑制剂Z-DEVD-FMK(默克,目录号:264155)
  17. Caspase-3底物Ac-DEVD-AMC(BD Biosciences,Pharmingen TM ,目录号:556449)
  18. 裂解缓冲液(见食谱)
  19. 分析缓冲液(见食谱)

设备

  1. 烤箱
  2. 发光分光光度计
  3. 移液器
  4. 涡流
  5. 离心分离机
  6. 平台振动筛
  7. 4°C冰箱
  8. -20°C冰柜
  9. 37°C培养箱

程序

  1. 蛋白质提取(图2A)
    1. 准备裂解缓冲液(见配方1)
    2. 在带有冰的培养皿中,放置载玻片并将涡虫转移到载玻片上。用剃刀刀片去除多余的水并将涡虫切成小块(必要时用一张纸去除剃刀刀片上的粘液)。
      注意:至少5个涡虫,每个条件3-5毫米。
    3. 每个涡虫添加100μl裂解缓冲液至载玻片,并用它将涡虫碎片吸入Eppendorf管中。把它放在冰上。 
    4. 通过用P200移液并涡旋5秒进行均质化,尽可能将Eppendorf管保持在冰上。
    5. 在4℃下以13,000 x g 离心10分钟。
    6. 将上清液部分转移至4℃的新管中。长期储存保存在-20°C。

  2. 使用Micro BCA Protein Assay Kit测定蛋白质浓度(图2B)
    1. 按照制造商的建议,进行必要的BSA蛋白稀释,以获得标准曲线。
    2. 按照制造商的建议准备工作试剂。
    3. 将样品稀释250倍(例如,2μl样品+498μlMilliQ水)。
    4. 向每个分光光度计比色皿中加入500μl工作试剂+500μl稀释样品。还应对用于获得标准曲线的样品进行该程序。
    5. 将分光光度计比色皿在60℃的烘箱中孵育1小时。 
    6. 从烘箱中取出样品,用分光光度计测量吸光度(λ= 562 nm)。
      注意:所有样品应在不超过10分钟的时间内读取。
    7. 使用步骤B1中进行的蛋白质稀释的值构建标准曲线。减去空白样品的值(没有蛋白质,在步骤B1中获得)以避免背景测量。计算样品的蛋白质浓度。将稀释因子的值乘以。

  3. Caspase-3活性测量(图2C)
    1. 准备化验缓冲液(见配方2)
    2. 装入96孔板。 
    3. 从空白井开始。空白溶液由125μl测定缓冲液+25μl裂解缓冲液组成。空白井必须一式三份完成。
      注意:96孔板中加载的所有样品必须一式三份。这意味着对于每个条件(空白,阴性对照和样品),您必须至少有三个孔。
    4. 加载实验阴性对照。对于阴性对照,应使用来自对照样品的蛋白质。在每个孔中加载123μl测定缓冲液+20μg蛋白质(xμl蛋白质提取物+yμl裂解缓冲液直至25μl体积)+1.5μlCaspase-3抑制剂Z-DEVD-FMK。阴性对照必须一式三份。
      注意:实验控制用于确认您正在测量Caspase-3活性。使用Caspase-3抑制剂被认为可以获得低水平的Caspase-3活性。必须有一个控件,但可以包含所需数量的控件。
    5. 在剩余的孔中加载123μl测定缓冲液+20μg蛋白质(xμl蛋白质提取物+yμl裂解缓冲液至25μl终体积)。所有样品必须一式三份进行。
    6. 将板在黑暗中于37℃孵育15分钟,同时搅拌。
    7. 将2μlCaspase-3底物Ac-DEVD-AMC加载到所有平板孔中,并在黑暗中于37℃孵育2小时。
    8. 使用发光分光光度计(激发380nm,发射440nm)通过发光测量酶活性。


    图2. Caspase-3检测方案的一般视图。 A.卡通示例蛋白质提取。 B.蛋白质浓度测定的逐步进行。 C.如何装载板孔的例子。

数据分析

  1. 平均三次技术重复以获得每个样品的单次读数。
  2. 减去空白样本的值以避免背景读取。
  3. 绘制阴性对照,实验对照和目标样品。

第二部分:TUNEL分析

末端脱氧核苷酸转移酶(TdT)dUTP缺口末端标记(TUNEL)测定已被设计用于检测呈现广泛DNA降解的凋亡细胞。该方法基于TdT标记独立于模板的双链DNA断裂的平末端的能力。

材料和试剂

  1. 玻璃瓶(Sigma-Aldrich,目录号:27151)
  2. 96孔板(VWR,目录号:734-2781)
  3. 载玻片(VWR,目录号:631-1551)
  4. 盖玻片(默克,目录号:S7117)
  5. ApopTag Red原位凋亡检测试剂盒(默克,目录号:S7165)
  6. 10%正乙酰半胱氨酸(用PBS 1x稀释)(Sigma-Aldrich,目录号:A9165)
  7. PBS 1x
  8. 37%甲醛(默克,目录号:104003)
  9. 1%SDS(用PBS 1x稀释)(Sigma-Aldrich,目录号:L3771)
  10. 30%H 2 O 2 (默克,目录号:107209)
  11. 漂白液
  12. TdT酶(来自ApopTag Kit)
  13. 抗洋地黄毒苷 - 罗丹明(来自ApopTag Kit)
  14. 70%甘油(用PBS 1x稀释)
  15. PBSTx(见食谱)
  16. PBSTB(见食谱)

设备

  1. 平台振动筛
  2. 移液器
  3. 37°C培养箱
  4. 4°C冰箱
  5. 共聚焦显微镜

软件

  1. ImageJ的

程序

  1. 将动物放入玻璃瓶中,用移液管移除涡虫水。
    注意:动物应该在3毫米到5毫米之间。
  2. 将动物与5ml 10%N-乙酰半胱氨酸(在PBS中1x稀释)在室温下孵育5分钟,摇动。
    注意: 
    1. 虽然玻璃瓶正在摇摆,但要确保涡虫正确分离并与溶液接触。
    2. 这一步杀死了动物并去除了厚厚的粘液涂层。
  3. 去除N-乙酰半胱氨酸,并在室温下用5毫升4%甲醛(用PBSTx稀释)固定动物20分钟,摇晃。
    注意:每次换液都应该通过移液小心进行,但不要接触带有尖端的涡虫,以避免组织损伤。
  4. 除去固定剂并用5ml 1%SDS(在PBS中1x稀释)在室温下孵育动物20分钟,摇动。
  5. 除去先前的溶液,并在室温下用5ml PBSTx洗涤动物两次,每次5分钟,摇动。
  6. 取出洗涤液,用5ml 6%H 2 O 2 (在PBSTx中稀释)过夜,在白光下过夜,不摇摆。
    注意:一夜之间大约16小时。请务必执行此漂白时间。
  7. 取出漂白液,在室温下用5毫升PBSTx洗涤动物两次,每次5分钟,摇匀。
    注意:在此步骤中,动物可以在4°C下储存几天(不超过3天)。
  8. 取出洗涤液,用5ml PBS 1x冲洗动物。
  9. 小心地将动物转移到96孔板上。
    注意:确保井中的所有动物都与溶液接触非常重要。如果您有几种动物,最好将它们分配到两到三个井中。
  10. 使用移液管移除PBS 1x并将动物与100μl在反应缓冲液中稀释的TdT酶(均来自ApopTag试剂盒)在37℃下孵育4小时,摇动。
  11. 去除TdT酶并用100μl终止/洗涤缓冲液(来自ApopTag试剂盒)冲洗动物。 
  12. 除去先前的溶液并用100μlPBSTB(PBSTx与0.25%BSA)在室温下洗涤动物两次,摇动5分钟。
  13. 取出洗涤液并用阻断溶液(均来自ApopTag试剂盒)稀释的100μl抗地高辛 - 罗丹明在室温下,在黑暗,摇摆下染色动物。
    注意:从这个步骤到方案结束,动物应该保持在黑暗中。
  14. 除去先前的溶液并用100μl的PBSTB洗涤染色的动物4次,每次在室温下摇动10分钟。继续在4℃下在PBSTB中洗涤过夜,摇摆。
  15. 洗涤动物,直到在室温下用PBSTB获得清晰的信号(图3),摇动。
    注意:每20/30分钟更换一次解决方案。
  16. 除去先前的溶液并向动物中加入70%甘油。
  17. 将动物转移到载玻片上并用盖玻片安装。必要时加入甘油。
    注意:为避免甘油蒸发和样品中断,请在盖玻片周围涂上透明指甲油。请注意不要在动物身上涂上清漆。


    图3. TUNEL检测的结果图像显示通过共聚焦显微镜成像的涡虫的尾部。白点对应于垂死细胞的细胞核。比例尺=250μm。

数据分析

  1. 使用共聚焦显微镜获取图像。 
  2. 分析ImageJ软件中的图像。

食谱

  1. 裂解缓冲液
    Tris-HCl(pH 8)5mM
    EDTA(pH 8)20 mM
    特里顿0.5%
    在冰上保留裂解缓冲液直至必要
  2. 分析缓冲区
    HEPES(pH 7.5)20 mM
    甘油10%
    DTT 2 mM
    在室温下保留测定缓冲液直至必要
  3. PBSTx
    含有0.3%Triton的1x PBS
  4. PBSTB
    PBSTx含0.25%BSA

致谢

这项工作得到了补助金BFU2008-01544和BFU2014-56055-P(MinisteriodeEducaciónyCiencia)和2009SGR1018(AGAUR)的资助。 N.S。得到了巴塞罗那大学APIF奖学金的支持。 Caspase-3活性测量和TUNEL测定方案分别改编自González-Estévez等人(2007)和Pellettieri 等人(2010)。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Almuedo-Castillo,M.,Crespo-Yanez,X.,Seebeck,F.,Bartscherer,K.,Salo,E。和Adell,T。(2014)。 JNK控制涡虫干细胞有丝分裂的发生,并引发再生和重塑所需的凋亡细胞死亡。 PLoS Genet 10(6):e1004400。
  2. Baguñá,J。和Romero,R。(1981)。 对涡虫中的生长,去除和再生过程中细胞类型的定量分析 Dugesia mediterranea 和 Dugesia tigrina 。 Hydrobiologia 84:181-194。
  3. de Sousa,N。(2017)。博士论文。 Hippo通路在涡虫中的作用。巴塞罗那大学。
  4. González-Estévez,C。和Salo,E。(2010)。 涡虫中的自噬和细胞凋亡。 细胞凋亡 15(3 ):279-292。
  5. González-Estévez,C.,Felix,D.A.,Aboobaker,A.A。和Salo,E。(2007)。 Gtdap-1 促进自噬,是再生过程中涡虫重塑的必要条件。饥饿。 Proc Natl Acad Sci U S A 104(33):13373-13378。
  6. González-Estévez,C.,Felix,D.A.,Rodriguez-Esteban,G。和Aboobaker,A.A。(2012)。 在饥饿引起的涡虫增生过程中,新生母细胞后代减少并增加细胞死亡。 Int J Dev Biol 56(1-3):83-91。
  7. Miller,C.M。和Newmark,P.A。(2012)。 胰岛素样肽可调节涡虫中的大小和成体干细胞。 Int J Dev Biol 56(1-3):75-82。
  8. Pellettieri,J。和Sanchez Alvarado,A。(2007)。 细胞更新和成人组织稳态:从人类到涡虫。 Annu Rev Genet 41:83-105。
  9. Pellettieri,J.,Fitzgerald,P.,Watanabe,S.,Mancuso,J.,Green,D.R。和Sanchez Alvarado,A。(2010)。 在涡虫再生过程中细胞死亡和组织重塑。 Dev Biol 338(1):76-85。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sousa, N. D. and Adell, T. (2018). Detection of Cell Death in Planarians. Bio-protocol 8(19): e3039. DOI: 10.21769/BioProtoc.3039.
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