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Acute Live/Dead Assay for the Analysis of Toxic Effects of Drugs on Cultured Neurons
急性存活/死亡实验分析药物对培养神经元的毒性作用   

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Abstract

The primary culture of central nervous system (CNS) neurons is a popular test system for a rapid, quantitative and reliable assessment of the effects of drugs on central neurons. Consequently, studies on the excitotoxicity of NMDA activation and on intracellular calcium handling machineries with respect to ischemic damage to the brain as well as neurodegenerative diseases have been highly productive (Ankarcrona et al., 1995). This created the need to establish a standard method for assessment of neurotoxicity. Several methods are currently being used, including LDH leakage and MTT assays (Mosmann, 1983; Decker and Lohmann-Matthes, 1988). We have used another common method for assessing acute cell death, the dead/live assay (Slepian et al., 1996). It provides a precise time and concentration evaluation of the process of cell death following exposure to a toxic substance, in our case, zeta-inhibitory peptide (ZIP), previously proposed to act as a selective PKM-zeta antagonist (Ling et al., 2002; Pastalkova et al., 2006; Sadeh et al., 2015). In this assay, we load cells with Calcein-AM, which, upon penetration into live neurons, is converted from a non-fluorescent compound into a highly fluorescent green fluorophore. Subsequently, we expose the neurons to different concentrations of ZIP for various durations, in the presence of propidium iodide (PI) which penetrates dead cells, and count red/green fluorescent cells. This method allows us to examine which cells were alive before, and died after exposure to the toxic substance as well as the time course of cell death.

Keywords: Cultured hippocampal neuron(体外培养海马神经元), Zeta inhibitory peptide(Zeta抑制肽), Live/dead imaging(活/死成像), Calcein(钙黄绿素), Propidium iodide(碘化丙锭)

Materials and Reagents

  1. 24 well plates (Nunc)
  2. Rat pups age P0, hippocampus (Figure 1)
  3. L-15 (Thermo Fisher Scientific, Gibco®, catalog number: 21083 )
  4. Glucose (Sigma-Aldrich)
  5. Gentamicin (Sigma-Aldrich, catalog number: G1272 )
  6. Trypsine (Thermo Fisher Scientific, Gibco®, catalog number: 150900046 )
  7. DNase
  8. Enriched MEM (Minimal Essential Medium, enriched with glucose (0.6%), Glutamax (2 mM), and gentamicin (15 μg/ml) (Thermo Fisher Scientific, Gibco®, catalog number: 32360-026 )
  9. B-27 (Thermo Fisher Scientific, Gibco®, catalog number: 17504 )
  10. Horse serum (Thermo Fisher Scientific, Gibco®, catalog number: 16050 )
  11. Fetal calf serum (Gibco)
  12. 5-Fluoro-2-deoxyuridine (FUDR) (Sigma-Aldrich, catalog number: F0503 )
  13. Calcein-AM (Sigma-Aldrich)
  14. Zeta inhibitory peptide (ZIP) (AnaSpec)
  15. Scr-ZIP (Anaspec, same amino acids as ZIP, in a scrambled order)
  16. Propidium iodide (Sigma-Aldrich)
  17. NaCl (Sigma-Aldrich)
  18. KCl
  19. MgCl2 (Sigma-Aldrich)
  20. CaCl2 (Sigma-Aldrich)
  21. HEPES (Sigma-Aldrich, catalog number: 83264 )
  22. Standard extracellular medium (see Recipes)


    Figure 1. 3D reconstruction of a rodent brain. In green-bilateral hippocampus (reproduced from Allen Brain Atlas)

Equipment

  1. Biological hood (Haroshet Ltd.)
  2. 37 °C/5% CO2 humidified incubator (Tuttnauer, model: TUTT2424-2 )
  3. Confocal laser scanning inverted microscope (Zeiss, model: LSM 510 ), with a 40x oil-immersion objective (1.3 NA)
  4. Standard desktop centrifuge
  5. Inverted microscope (Nikon) equipped with phase optics
  6. Dissecting binocular microscope

Software

  1. ImageJ
  2. LSM 510 software

Procedure

  1. Primary brain cultures
    1. Wistar Rat pups are decapitated on day of birth (P0), their brains removed and placed in a chilled (4 °C), oxygenated (by bubbling with a pipette tip) Leibovitz L15 medium enriched with 0.6% glucose and gentamicin (20 µg/ml) under a dissecting microscope. The hippocampus is removed and the meninges are carefully peeled off.
    2. Hippocampal tissue is mechanically dissociated by trituration after incubation with trypsin (0.25%) and DNase (50 µg/ml), for 20 min at 37 °C.
    3. 37 °C 5% CO2, in a humidified incubator. The tissue is washed 3x with 2 ml of the L15 medium by centrifugation and passed to the plating medium. This consists of 1 ml minimum essential medium (MEM, Earl salts) containing 5% heat-inactivated horse serum (HS), 5% fetal calf serum, and B-27 (1 µl/1ml), enriched with 0.6% glucose, gentamicin (20 µg/ml), and 2 mM glutamax (enriched MEM). Cell number can either be determined by counting trypan blue-stained cells on a Neurobauer (Boeco, Germany), or estimated, assuming that each hippocampus contains appx. 1 million cells. About 105 cells in 1 ml medium are plated in each well of a 24-well plate, onto a hippocampal glial feeder layer which is grown on the glass for 2 weeks prior to the plating of the neurons (Papa et al., 1995, Goldin et al., 2001).
    4. Cells are left to grow in the humidified incubator at 37 °C, 5% CO2 for 4 days, at which time the medium is changed to 10% HS in enriched MEM, plus a mixture of 5´-fluoro-2-deoxyuridine/uridine (FUDR) (20 µg and 50 µg/ml, respectively), to block glial proliferation. Four days later the medium is replaced by 1ml 10% HS in MEM which does not contain FUDR, and no further changes are made.
      Note: We routinely use Wistar rats, but the procedure should be applicable to other rat strains and with small modifications, also to mouse cultures. The procedure is similar in other brain areas, and the neocortex or striatum have been used successfully using this method. We did not try cerebellar or spinal cord cultures. We use P0 rat pups, and this protocol is useful until P4-5, beyond that the yield of viable cells is reduced dramatically.

  2. Viability assay
    1. Cells are incubated in standard extracellular medium at room temperature and imaged on a stage of a Zeiss LSM 510 laser scanning confocal microscope, with a 40x oil-immersion objective (1.3 NA).
    2. Cultures are initially loaded in 1 ml standard recording medium with 2 µM Calcein-AM for 30 min, and imaged in 1.8 ml of standard medium in the presence of PI (2.5 µM). A new vile of ZIP or Scr-ZIP (a scrambled peptide used as control) was used each day, diluted to a final concentration of 1mM and kept at -4 °C, or control medium are added at 5-10 min after onset of imaging.
    3. Two-channel images [Calcein-AM/Propidium Iodide (PI)] are acquired, where green (488 nm) and red (545 nm) fluorescent cells represent live and dead cells, respectively.
    4. Time-lapse imaging of cultures is performed at room temperature for 90 min at 4 min intervals. Several fields of view per coverslip are imaged repeatedly. Survival probability is calculated as [[no. live cells (cells stained green) (attime t)/no. live cells (t = 0)]*100]. Cells that either lose their color (Calcein-AM leaked out) or gain red staining (PI entering the cell) were not regarded as live (Figure 2). No co-staining of red and green is detected. Neurons can easily be distinguished from glial cells, in that the latter group consists of large flat cells, whereas the neuronal somata are more compact and 3-dimensional.
    5. Standard imaging software (ImageJ) is used to count the number of live cells for each image.


      Figure 2. Measuring viability of hippocampal cultures in response to ZIP application. A. Viability of cultured hippocampal neurons was determined using Calcein-AM (green) and propidium iodide (red). Cultures were exposed to different doses of ZIP [Vehicle (0 µM)/1 µM/2 µM/5 µM/10 µM]. Scale bar, 50 µm; B. Dose response curve was constructed from time-lapse images taken in 4 min intervals (Figure 3) to assess the rate of cellular death following treatment with ZIP; C. Bar graph summarizes total change in culture viability (n = 22 coverslips, 3,544 cells, values are mean ± SEM, 90 min post ZIP-treatment vs. viability pre-treatment, paired t-test, ***P < 0.005); D. Scr-ZIP induces similar changes in culture viability. Bar graphs summarize total change in culture viability (n = 13 coverslips, 2,365 cells, 90 min post scr-ZIP-treatment vs. viability pre-treatment, paired t-test, ***P < 0.005). (Adapted from Sadeh et al., 2015)


      Figure 3. Illustration of time-lapse imaging of hippocampal cultures. Cells were loaded with Calcein-AM and Imaged in the presence of propidium iodide. 10 μM ZIP was added to the culture at t = 0. A cell was considered alive in the cell count as long as it retained green fluorescence.

Notes

  1. The method allows us to distinguish between cells that were already dead before the onset of drug treatment (those stained with PI but not stained earlier with Calcein-AM), those that died during and after the treatment (initially stained with Calcein-AM) and those which survived the damage (stained only with Calcein-AM).
  2. In addition the procedure allows distinguishing between neurons and glia, based on their different morphology.
  3. Additional stain for glia is possible with the use of selective live glia stains (Nimmerjahn et al., 2004).
  4. Finally, the time-lapse imaging of the cells allows to trace the course of cell death in the neuronal population (e.g., Figure 3).

Recipes

  1. Standard extracellular medium (filtered and stored in 0-4 °C refrigerator for up to 1 mo.)
    pH = 7.4, 32 mOsm
    129 mM NaCl
    4 mM KCl
    1 mM MgCl2
    2 mM CaCl2
    10.5 mM glucose
    10 mM HEPES

Acknowledgments

Adapted from “Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase Mζ, Is Excitotoxic to Cultured Hippocampal Neurons” (Sadeh et al., 2015).

References

  1. Ankarcrona, M., Dypbukt, J. M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S. A. and Nicotera, P. (1995). Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15(4): 961-973.
  2. Decker, T. and Lohmann-Matthes, M. L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 115(1): 61-69.
  3. Goldin, M., Segal, M. and Avignone, E. (2001). Functional plasticity triggers formation and pruning of dendritic spines in cultured hippocampal networks. J Neurosci 21(1): 186-193.
  4. Ling, D. S., Benardo, L. S., Serrano, P. A., Blace, N., Kelly, M. T., Crary, J. F. and Sacktor, T. C. (2002). Protein kinase Mzeta is necessary and sufficient for LTP maintenance. Nat Neurosci 5(4): 295-296.
  5. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1-2): 55-63.
  6. Papa, M., Bundman, M. C., Greenberger, V. and Segal, M. (1995). Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons. J Neurosci 15(1 Pt 1): 1-11.
  7. Pastalkova, E., Serrano, P., Pinkhasova, D., Wallace, E., Fenton, A. A. and Sacktor, T. C. (2006). Storage of spatial information by the maintenance mechanism of LTP. Science 313(5790): 1141-1144.
  8. Sadeh, N., Verbitsky, S., Dudai, Y. and Segal, M. (2015). Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase Mzeta, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci 35(36): 12404-12411.
  9. Slepian, M. J., Massia, S. P. and Whitesell, L. (1996). Pre-conditioning of smooth muscle cells via induction of the heat shock response limits proliferation following mechanical injury. Biochem Biophys Res Commun 225(2): 600-607.

简介

中枢神经系统(CNS)神经元的主要培养物是用于快速,定量和可靠地评估药物对中枢神经元的影响的流行测试系统。因此,关于NMDA激活和细胞内钙处理机制对脑缺血性损伤以及神经退行性疾病的兴奋性毒性的研究已经很有成效(Ankarcrona等,1995)。这就需要建立一种评估神经毒性的标准方法。目前正在使用几种方法,包括LDH渗漏和MTT测定(Mosmann,1983; Decker和Lohmann-Matthes,1988)。我们使用另一种常见的方法来评估急性细胞死亡,死亡/活体检测(Slepian等,1996)。它提供了暴露于有毒物质后细胞死亡过程的精确时间和浓度评估,在我们的情况下,以前提出用作选择性PKM-zeta拮抗剂的ζ抑制肽(ZIP)(Ling等, 2002; Pastalkova等人,2006; Sadeh等人,2015)。在该测定中,我们用Calcein-AM负载细胞,其在穿透到活神经元中时,从非荧光化合物转化为高度荧光的绿色荧光团。随后,在渗透死细胞的碘化丙啶(PI)和计数红/绿荧光细胞的存在下,我们将神经元暴露于不同浓度的ZIP各种持续时间。这种方法可以让我们检查哪些细胞是活的,暴露在有毒物质之后死亡,以及细胞死亡的时间过程。

关键字:体外培养海马神经元, Zeta抑制肽, 活/死成像, 钙黄绿素, 碘化丙锭

材料和试剂

  1. 24孔板(Nunc)
  2. 大鼠幼年P0,海马(图1)
  3. L-15(Thermo Fisher Scientific,Gibco ,目录号:21083)
  4. 葡萄糖(Sigma-Aldrich)
  5. 庆大霉素(Sigma-Aldrich,目录号:G1272)
  6. Trypsine(Thermo Fisher Scientific,Gibco ,目录号:150900046)
  7. DNase
  8. 富集的富含葡萄糖(0.6%),Glutamax(2mM)和庆大霉素(15μg/ml)(Gibco,目录号:32360-026)的MEM(最低必需培养基)
  9. B-27(Thermo Fisher Scientific,Gibco ,目录号:17504)
  10. 马血清(Thermo Fisher Scientific,Gibco ,目录号:16050)
  11. 胎牛血清(Gibco)
  12. 5-氟-2-脱氧尿苷(FUDR)(Sigma-Aldrich,目录号:F0503)
  13. Calcein-AM(Sigma-Aldrich)
  14. Zeta抑制肽(ZIP)(AnaSpec)
  15. Scr-ZIP(Anaspec,与ZIP相同的氨基酸,以乱序排序)
  16. 碘化丙啶(Sigma-Aldrich)
  17. NaCl(Sigma-Aldrich)
  18. KCl
  19. MgCl 2(Sigma-Aldrich)
  20. CaCl 2(Sigma-Aldrich)
  21. HEPES(Sigma-Aldrich,目录号:83264)
  22. 标准细胞外介质(参见配方)


    图1.啮齿动物脑的3D重建。在绿色 - 双侧海马(由Allen Brain Atlas翻译)

设备

  1. 生物罩(Haroshet Ltd.)
  2. 37℃/5%CO 2加湿培养箱(Tuttnauer,型号:TUTT2424-2)
  3. 共聚焦激光扫描倒置显微镜(Zeiss,型号:LSM 510),具有40x油浸物镜(1.3NA)
  4. 标准台式离心机
  5. 装有相位光学装置的倒置显微镜(尼康)
  6. 解剖双目显微镜

软件

  1. 图像J
  2. LSM 510软件

程序

  1. 原发性脑培养
    1. 将Wistar Rat幼鼠在出生当天断头(P0),取出它们的大脑并放置在冷冻(4℃),充氧(通过用移液管吸头鼓泡)的Leibovitz L15培养基,富含0.6%葡萄糖和庆大霉素(20μg/ml)。除去海马并小心地剥离脑膜。
    2. 通过在与胰蛋白酶(0.25%)和DNase(50μg/ml)孵育后在37℃下孵育20分钟,通过研磨机械解离海马组织。
    3. 37℃,5%CO 2中。通过离心将组织用2ml的L15培养基洗涤3次,并传递至平板培养基。其由含有5%热灭活的马血清(HS),5%胎牛血清和富含0.6%葡萄糖的B-27(1μl/1ml)的1ml最小必需培养基(MEM,Earl盐),庆大霉素(20μg/ml)和2mM glu??tamax(富集的MEM)。细胞数可以通过在Neurobauer(Boeco,Germany)上计数台盼蓝染色的细胞来测定,或估计,假设每个海马含有大肠杆菌。 1百万细胞。将1ml培养基中的约10 5个细胞接种在24孔板的每个孔中,接种到海马神经胶质饲养层上,所述海马神经胶质饲养层在玻璃上生长2周,然后铺板神经元( Papa等人,1995,Goldin等人,2001)。
    4. 使细胞在湿润的培养箱中在37℃,5%CO 2下生长4天,此时将培养基变为富含MEM中的10%HS,加上5% - 氟-2-脱氧尿苷/尿苷(FUDR)(分别为20μg和50μg/ml),以阻断胶质增生。 4天后,用不含FUDR的MEM中的1ml 10%HS替换培养基,并且不进行进一步的改变。
      注意:我们常规使用Wistar大鼠,但是该程序应该适用于其他大鼠品系,并且具有小的修饰,也适用于小鼠培养物。该程序在其他大脑区域是类似的,并且新皮质或纹状体已成功使用这种方法。我们没有尝试小脑或脊髓文化。我们使用P0大鼠,这个协议是有用的,直到P4-5,超出活细胞的产量大大减少。

  2. 活力测定
    1. 将细胞在室温下在标准细胞外培养基中温育,并在具有40x油浸物镜(1.3NA)的Zeiss LSM 510激光扫描共聚焦显微镜的台上成像。
    2. 首先将培养物加载到具有2μM钙荧光素-AM的1ml标准记录介质中30分钟,并在PI(2.5μM)存在下在1.8ml标准培养基中成像。每天使用新的ZIP或Scr-ZIP(用作对照的乱序肽),稀释至1mM的终浓度并保持在-4℃,或在起始后5-10分钟加入对照培养基成像。
    3. 获得两通道图像[Calcein-AM /碘化丙啶(PI)],其中绿色(488nm)和红色(545nm)荧光细胞分别代表活细胞和死细胞。
    4. 培养物的时间推移成像在室温下以4分钟间隔进行90分钟。每个盖玻片的几个视场被重复成像。生存概率计算为[[no。活细胞(细胞染成绿色)(attime t)/无。活细胞(t = 0)] * 100]。失去其颜色(钙黄绿素-AM泄漏出)或获得红色染色(PI进入细胞)的细胞不被认为是活的(图2)。没有检测到红色和绿色的共染色。神经元可以容易地与神经胶质细胞区分,因为后者组由大的扁平细胞组成,而神经元胞体更紧凑和3维。
    5. 标准成像软件(Image J)用于计数每个图像的活细胞数

      图2.测量应答ZIP应用的海马培养物的生存力。A.使用钙荧光素-AM(绿色)和碘化丙啶(红色)测定培养的海马神经元的活力。将培养物暴露于不同剂量的ZIP [载体(0μM)/1μM/2μM/5μM/10μM]。比例尺,50μm; B.从以4分钟间隔获取的时间推移图像构建剂量反应曲线(图3),以评估用ZIP处理后的细胞死亡率; C.柱状图总结了培养物存活力的总变化(n = 22盖玻片,3,544个细胞,值是平均值±SEM,在ZIP处理后90分钟对比活力预处理,配对t检验,*** < <0.005); D.Scr-ZIP诱导培养物生存力的相似变化。条形图总结了培养物存活力的总变化(n = 13盖玻片,2,365个细胞,scr-ZIP-处理后90分钟与存活力预处理,配对t-检验,*** p < 0.005)。 (摘自Sadeh等人,2015年)。


      图3.海马培养物时间推移成像的图示。细胞加载钙黄绿素-AM并在碘化丙啶存在下成像。在t = 0时向培养物中加入10μMZIP。细胞被认为在细胞计数中存活,只要它保持绿色荧光。

笔记

  1. 该方法允许我们区分在药物治疗开始之前已经死亡的细胞(用PI染色但是没有用钙黄绿素-AM早期染色的细胞),在治疗期间和之后死亡的细胞(最初用钙黄绿素-AM染色)和那些幸存的损伤(仅用钙黄绿素AM染色)。
  2. 此外,该程序允许基于其不同的形态在神经元和神经胶质之间进行区分。
  3. 使用选择性活胶质污渍可以使胶质的其它染色成为可能(Nimmerjahn等人,2004)。
  4. 最后,细胞的时间推移成像允许追踪神经元群体中细胞死亡的过程(例如,图3)。

食谱

  1. 标准细胞外培养基(过滤并在0-4℃冰箱中储存长达1个月) pH = 7.4,32mOsm
    129 mM NaCl 4 mM KCl
    1mM MgCl 2
    2mM CaCl 2 2 / 10.5 mM葡萄糖 10 mM HEPES

致谢

改编自"Zeta抑制肽,蛋白激酶Mζ的候选抑制剂,对培养海马神经元有兴趣毒性"(Sadeh等人,2015)。

参考文献

  1. Anfarcrona,M.,Dypbukt,JM,Bonfoco,E.,Zhivotovsky,B.,Orrenius,S.,Lipton,SA和Nicotera,P。(1995)。  谷氨酸诱导的神经元死亡:依赖于线粒体功能的一系列坏死或凋亡。神经元 15(4):961-973。
  2. Decker,T.和Lohmann-Matthes,ML(1988)。 
  3. Goldin,M.,Segal,M。和Avignone,E。(2001)。  功能可塑性触发树突棘在培养的海马网络中的形成和修剪。 J Neurosci 21(1):186-193。
  4. Ling,DS,Benardo,LS,Serrano,PA,Blace,N.,Kelly,MT,Crary,JF and Sacktor,TC(2002)。  蛋白激酶Mzeta对于LTP维持是必要的和充分的。 Nat Neurosci 5(4):295 -296。
  5. Mosmann,T。(1983)。  快速比色法细胞生长和存活:应用于增殖和细胞毒性测定。 Immunol Methods 65(1-2):55-63。
  6. Papa,M.,Bundman,MC,Greenberger,V.and Segal,M。(1995)。  通过LTP的维护机制存储空间信息。 313(5790):1141-1144 。
  7. Sadeh,N.,Verbitsky,S.,Dudai,Y.和Segal,M.(2015)。  Zeta Inhibitory Peptide,Candidate Inhibitor of Protein Kinase Mizeta,Is Excitotoxic to Cultured Hippocampal Neurons。
  8. Slepian,MJ,Massia,SP和Whitesell,L.(1996)。  通过诱导热休克反应预处理平滑肌细胞限制机械损伤后的增殖。生物化学生物物理研究通讯(Biochem Biophys Res Commun)225(2):600-607。
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Copyright: © 2016 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. Sadeh, N., Oni-Biton, E. and Segal, M. (2016). Acute Live/Dead Assay for the Analysis of Toxic Effects of Drugs on Cultured Neurons. Bio-protocol 6(15): e1889. DOI: 10.21769/BioProtoc.1889.
  2. Sadeh, N., Verbitsky, S., Dudai, Y. and Segal, M. (2015). Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase Mzeta, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci 35(36): 12404-12411.
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