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Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, many of its neurons can be exclusively targeted using cell-specific promoters. We have recently taken advantage of optogentics to deliver artificial patterns of prolonged activation to a class of mechanosensory neurons, called touch receptor neurons (TRNs) in worms that lack touch sensation due to a genetic mutation. Our aim was to examine whether we can counteract the effects of sensory loss by artificially activating the sensory neurons. Here we describe in detail the various components of the protocol that we used. This consists of exposing worms expressing the light-sensitive ion channel Channelrohdopsin 2 (ChR2) in TRNs to long-term random flashes of light.

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Artificial Optogenetic TRN Stimulation of C. elegans
人工光基因刺激线虫触觉神经实验

神经科学 > 行为神经科学 > 实验动物模型 > 其它
作者: Ithai Rabinowitch
Ithai RabinowitchAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
For correspondence: ithairab@gmail.com
Bio-protocol author page: a3605
Millet Treinin
Millet TreininAffiliation: Department of Medical Neurobiology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3606
 and Jihong Bai
Jihong BaiAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
Bio-protocol author page: a3607
Vol 6, Iss 20, 10/20/2016, 929 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1966

[Abstract] Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, many of its neurons can be exclusively targeted using cell-specific promoters. We have recently taken advantage of optogentics to deliver artificial patterns of prolonged activation to a class of mechanosensory neurons, called touch receptor neurons (TRNs) in worms that lack touch sensation due to a genetic mutation. Our aim was to examine whether we can counteract the effects of sensory loss by artificially activating the sensory neurons. Here we describe in detail the various components of the protocol that we used. This consists of exposing worms expressing the light-sensitive ion channel Channelrohdopsin 2 (ChR2) in TRNs to long-term random flashes of light.
Keywords: Optogenetics(光遗传学), C. elegans(线虫), Mechanosensation(机械感受), Locomotion(运动), Cross-modal plasticity(跨模态可塑性)

[Background] Artificial optogenetic stimulation (or silencing) of neurons has become of broad use in neuroscientific research. The powerful model organism, C. elegans, is particularly amenable to optogenetic manipulation (Nagel et al., 2005), and multiple groups have developed a range of techniques for delivering artificial brief patterns of stimulation with high temporal and spatial precision (Leifer et al., 2011; Stirman et al., 2011) and in combination with behavioral (Kocabas et al., 2012) and calcium imaging (Guo et al., 2009) or electrophysiological (Lindsay et al., 2011) readouts. We were interested in establishing a long-term stimulation protocol that would substitute natural ongoing activity in mechanosensory neurons deprived of sensory input (Rabinowitch et al., 2016). Our protocol integrates previous C. elegans optogenetic protocols, but focuses on chronic rather than transient stimulation.

[Abstract]

Materials and Reagents

  1. 1.7 ml microtubes (Genesee Scientific, catalog number: 24-282 )
  2. Aluminum foil
  3. 60 x 15 mm cultivation/test plates (Genesee Scientific, catalog number: 32-105G )
  4. 35 x 10 mm stimulation plates (Genesee Scientific, catalog number: 32-103 )
  5. C. elegans strains
    1. TU253 mec-4(u253) X (a mutant strain deficient in TRN mechanosensation). mec-4 encodes an amiloride-sensitive Na+ channel protein (degenerin) expressed exclusively in the TRNs and required to sense gentle mechanical stimuli (e.g., touch) along the body wall (www.wormbase.org)
    2. BJH255 mec-4(u253) X; ljIs111[Pmec-4::ChR2]
    Notes:
    1. The second strain combines both defective TRN mechanosensation and ChannelRhodopsin 2 (ChR2) expressed exclusively in the TRNs. Importantly, while examining several TRN-specific ChR2 strains (all using the mec-4 promoter), we observed in some of them abnormal mechanosensation and locomotion even in a wild-type background. In contrast, ljIs111 exhibited normal behavior and mechanosensory responses.
    2. Strains are grown and maintained under standard conditions (http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html) at 20 °C on nematode growth medium (NGM) 2% agar cultivation plates seeded with Escherichia coli strain OP50. All experiments are conducted at 18-22 °C. We found that higher temperatures considerably alter the results. Tested worms are adults around 24 h after the L4 stage.
    3. C. elegans does not produce the co-factor all-trans retinal (ATR), necessary for ChR2 function. However, ATR can be incorporated through feeding (Nagel et al., 2005). ATR is first diluted in ethanol to 100 mM in a 1.7 ml tube. The tube is wrapped with aluminum foil to avoid light exposure and is stored at -20 °C. Prior to seeding the ATR plates, 5 μl of the 100 mM ATR stock solution is added to 1 ml OP50 bacteria suspended in LB and the tube is gently vortexed. Then 100 μl of the ATR OP50 mix is applied to 6-cm cultivation plates and to 3-cm stimulation plates, each containing nematode growth medium (NGM). Once seeded, the plates are kept in the dark. The plates can be used the next day.
    4. ChR2-expressing worms are grown in the dark on the 6-cm ATR cultivation plates. As a control, a second cohort of worms can be grown under similar conditions, but without the ATR.
  6. Lysogeny broth medium (LB) (RPI, catalog number: L24045-5000.0 )
  7. Bacto agar (BD, catalog number: 214040 )
  8. Bacto peptone (BD, catalog number: 211820 )
  9. Sodium chloride (NaCl)
  10. Magnesium sulfate (MgSO4)
  11. Cholesterol
  12. Calcium chloride (CaCl2)
  13. All-trans retinal (ATR) (Sigma-Aldrich, catalog number: R2500-25MG )
  14. Ethanol (Decon Labs, catalog number: 2701 )
  15. Escherichia coli strain OP50 (see Recipes)
  16. Nematode growth medium (NGM) (see Recipes)

Equipment

  1. 4 L glass flask
  2. Incubator
  3. Royal-Blue (447.5 nm) LUXEON Rebel LED assembly (of 3 LEDs) (Luxeon Star LEDs, model: SR-03-R0500 )
  4. Carclo 27° frosted 20 mm circular beam optic (Luxeon Star LEDs, catalog number: 10508 )
  5. Arduino Uno R3 microcontroller (Adafruit, model: Arduino Uno R3)
  6. Personal computer (PC or Mac)
  7. USB 2.0 cable (SparkFun Electronics, model: CAB-00512 )
  8. Hook-up wire (Alpha Wire, catalog number: 2842/19 )
  9. Solder station (Apex Tool, Weller®, model: WLC100 )


    Figure 1. Optical stimulation apparatus. A. LED assembly and beam optic. White arrows point towards soldering points. B. LED assembly mounted to the top of an opaque cardboard box. C. Hook-up wires from the LED assembly (black arrowhead) connected to the digital output of an Arduino Uno microcontroller board. In the picture the Arduino board is not connected to the computer.

  10. Rosin core solder (Alpha Fry 31604 60/40)
  11. Opaque cardboard box (9 x 4.5 x 6 cm, length x width x height)
    Note: Two pieces of hook-up wire are soldered to the LED assembly to drive current through it. The LED assembly is attached to the circular beam optic and together they are mounted to the top of the opaque cardboard box. The LED is connected to an Arduino Uno R3 microcontroller. One wire is attached to the ground and the other to digital output number 12. Figure 1 illustrates the apparatus. The Arduino microcontroller is connected to the computer via USB cable.

Software

  1. Arduino software (https://www.arduino.cc)
    Note: Available for free online.
  2. Matlab support package for Arduino (Mathworks, http://www.mathworks.com/hardware-support/arduino-matlab.html)
    Note: Available for free online.
  3. Matlab 2014R (Mathworks, http://www.mathworks.com/products/matlab/)
  4. Custom written Matlab script called Led3.m, provided as an appendix.
    Notes:
    1. In the Led3.m script, configuration Poisson4 is used to deliver random flashes of blue light for approximately 80 min each session, producing prolonged stimulation.
    2. The interval between flashes is drawn from an exponential random distribution with a 10 sec mean. The duration of each flash is drawn from a uniform distribution with a 3 sec mean.

Procedure

  1. Prior to blue light stimulation, 10 worms are transferred from the 6-cm cultivation plate to a 3-cm seeded stimulation plate including ATR. The lid of the plate is removed (so that it does not interfere with the light) and the plate is placed inside the optical stimulation box. Then stimulation is started by running the Led3.m Matlab script.
  2. In our application, we were interested in examining the effects of prolonged artificial TRN stimulation on subsequent locomotion patterns of mechanosensory mutants. Since we found that loss of body mechanosensation leads to an enhanced reversing frequency when the worms are removed from their source of food, we tested reversing frequency after artificial TRN stimulation to see if it decreases back towards wild-type levels (Rabinowitch et al., 2016).
  3. In each testing round, four to six worms of each genotype are picked from the 3-cm stimulation plate either immediately, after no flashing at all or 2 h after the end of the flashing session, and their reversing rate off food is measured. The assay is repeated on at least three different testing days, and in each testing day all genotypes are equally represented.
  4. The reversing assay is based on a well-established protocol (e.g., Tsalik and Hobert, 2003). Each worm is first transferred to an area in the plate that is free of food in order to release bacterial remainders from its body. As soon as there is no visible trail of food, the worm is placed on a 6-cm unseeded NGM test plate. Prior to the assay, the test plate is left for 1 h with its lid off to dry its surface. 1 min after transferring the worm to the test plate, the number of reversal events is counted over a duration of 3 min. A reversal was counted only if it consisted of at least two consecutive head bends while the worm reversed.
    Note: We observed an effect only 2 h after the end of the light stimulation.

Data analysis

Each experiment is performed on at least 3 separate days, with a similar number of repetitions per day. Mean reversing frequency (number of reversals per minute) is calculated by dividing the number of reversal events by 3 min. In experiments in which a 2 x 2 design is applied (e.g., no stimulation versus stimulation and wild-type worms versus mechanosensory mutants), a 2 x 2 ANOVA is used to establish whether a significant interaction exists between the two categories. Post hoc t-tests with Bonferroni corrections are then used to compare within each category. Alternatively, reversing rate values are normalized by the average reversing rate without any stimulation for each experiment day and a t-test is used to compare between experimental conditions (e.g., wild-type versus mechanosensory mutants). Examples of raw data can be found in http://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.1002348#pbio.1002348.s001, which is a supplement to our original research paper (Rabinowitch et al., 2016).

Notes

  1. C. elegans behavior varies considerably as a function of its environmental conditions. We noticed that reversing rate might change dramatically from day to day. A key parameter affecting reversing rate was the dryness of the test plate. There was also differential variability in reversing between the mechanosensory mutants and wild-type worms. Here a key factor was temperature. In order to overcome this variation and cancel out its effects, each experiment is spread out over several testing days.

Recipes

  1. Escherichia coli strain OP50
    1. Prepare a bottle of 500 ml LB (according to product recipe).
    2. Inoculate the LB with a single colony of OP50.
    3. Close lid loosely and place in a 37 °C incubator.
    4. Grow overnight without shaking and the next day place in a 4 °C refrigerator.
  2. Nematode growth medium (NGM) (4 L)
    1. Mix well: 72 g Bacto agar; 10 g Bacto peptone and 12 g NaCl in 4 L glass flask.
    2. Add 3.8 L H2O. Place stir bar inside flask and autoclave for 60 min.
    3. Right after autoclave add: 4 ml 1 M MgSO4, 100 ml 1 M K-phosphate buffer, pH 6.0 and 4 ml 5 mg/ml cholesterol.
    4. Place flask in 55 °C and stir.
    5. After media has cooled add 4 ml 1 M CaCl2.
    6. While still warm pour 10 ml in each 6-cm plate.

Acknowledgments

Support for writing the protocol was provided by an FHCRC New Development Grant, and NIH grant NINDS R01NS085214. The current protocol was adapted from previous work (Nagel et al., 2005)

References

  1. Guo, Z. V., Hart, A. C. and Ramanathan, S. (2009). Optical interrogation of neural circuits in Caenorhabditis elegans. Nat Methods 6(12): 891-896.
  2. Kocabas, A., Shen, C. H., Guo, Z. V. and Ramanathan, S. (2012). Controlling interneuron activity in Caenorhabditis elegans to evoke chemotactic behaviour. Nature 490(7419): 273-277.
  3. Leifer, A. M., Fang-Yen, C., Gershow, M., Alkema, M. J. and Samuel, A. D. (2011). Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat Methods 8(2): 147-152.
  4. Lindsay, T. H., Thiele, T. R. and Lockery, S. R. (2011). Optogenetic analysis of synaptic transmission in the central nervous system of the nematode Caenorhabditis elegans. Nat Commun 2: 306.
  5. Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E. and Gottschalk, A. (2005). Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15(24): 2279-2284.
  6. Rabinowitch, I., Laurent, P., Zhao, B., Walker, D., Beets, I., Schoofs, L., Bai, J., Schafer, W. R. and Treinin, M. (2016). Neuropeptide-driven cross-modal plasticity following sensory loss in Caenorhabditis elegans. PLoS Biol 14(1): e1002348.
  7. Stirman, J. N., Crane, M. M., Husson, S. J., Wabnig, S., Schultheis, C., Gottschalk, A. and Lu, H. (2011). Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans. Nat Methods 8(2): 153-158.
  8. Tsalik, E. L. and Hobert, O. (2003). Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans. J Neurobiol 56: 178-197.

材料和试剂

  1. 1.7ml微管(Genesee Scientific,目录号:24-282)
  2. 铝箔
  3. 60×15mm培养/测试板(Genesee Scientific,目录号:32-105G)
  4. 35×10mm刺激板(Genesee Scientific,目录号:32-103)
  5. C。 elegans 菌株
    1. TU253mec-4(u253)X(TRN mechanosensation缺陷的突变株)。 mec-4 编码在TRNs中独占表达并且需要感测温和机械刺激的阿米洛利敏感性Na +通道蛋白(简并蛋白)(例如 。,触摸)沿着身体墙( www.wormbase.org
    2. BJH255 emec-4(u253)X; ljIs111 [Pmec-4 :: ChR2 ]
    注意:
    1. 第二应变结合缺陷TRN mechanosensation和ChannelRhodopsin 2(ChR2)专门表示在TRNs。重要的是,虽然检查几个TRN特定ChR2株(所有使用mec-4启动子),我们观察到其中一些异常mechanosensation和运动甚至在野生型背景。相反,ljIs111表现出正常的行为和机械感觉反应。
    2. 设备

      1. 4升玻璃瓶
      2. 孵化器
      3. Royal-Blue(447.5 nm)LUXEON Rebel LED组件(3个LED)(Luxeon Star LED,型号:SR-03-R0500)
      4. Carclo 27°磨砂20 mm圆形光束(Luxeon Star LED,目录号:10508)
      5. Arduino Uno R3微控制器(Adafruit,型号:Arduino Uno R3)
      6. 个人计算机(PC或Mac)
      7. USB 2.0电缆(SparkFun Electronics,型号:CAB-00512)
      8. 连接线(Alpha Wire,目录号:2842/19)
      9. 焊接站(Apex Tool,Weller ,型号:WLC100)


        图1.光学刺激装置 A. LED组件和光束光学元件。白色箭头指向焊点。 B. LED组件安装在不透明纸板箱的顶部。 C.来自LED组件的连接线(黑色箭头)连接到Arduino Uno微控制器板的数字输出。在图片中,Arduino板未连接到计算机。

      10. 松香芯焊料(Alpha Fry 31604 60/40)
      11. 不透明纸板箱(9 x 4.5 x 6厘米,长x宽x高)
        注意:两个连接线焊接到LED组件,以驱动电流通过它。 LED组件附接到圆形光束光学器件,并且它们一起安装到不透明纸板箱的顶部。 LED连接到Arduino Uno R3微控制器。一根线连接到地面,另一根连接到数字输出编号12.图1说明了该装置。 Arduino微控制器通过USB电缆连接到计算机。

      软件

      1. Arduino软件( https://www.arduino.cc
        注意:可免费在线。
      2. 用于Arduino的Matlab支持包(Mathworks, http://www.mathworks.com/hardware-support/arduino-matlab.html
        注意:可免费在线。
      3. Matlab 2014R(Mathworks, http://www.mathworks.com/products/matlab/
      4. 自定义的Matlab脚本Led3.m,提供作为附录 注意:
        1. 在Led3.m脚本中,配置 Poisson4 用于在每次会话中递送蓝光的随机闪烁约80分钟,产生延长的刺激。 em>
        2. 闪光之间的间隔是从具有10秒平均值的指数随机分布绘制的。每个闪光的持续时间来自具有3秒平均值的均匀分布。

      程序

      1. 在蓝光刺激之前,将10个蠕虫从6cm培养板转移到包括ATR的3cm播种刺激板。移除板的盖(使得其不干扰光),并且将板放置在光刺激箱内。然后通过运行Led3.m Matlab脚本启动刺激
      2. 在我们的应用程序,我们有兴趣检查延长的人工TRN刺激对随后的机械感觉突变体的运动模式的影响。由于我们发现当蠕虫从其食物来源移除时,身体机械损伤的损失导致增强的逆转频率,所以我们在人工TRN刺激后测试逆转频率,以观察其是否降回到野生型水平(Rabinowitch等人al 。,2016)。
      3. 在每个测试轮中,在闪光阶段结束后根本不闪烁或2小时后立即从3cm刺激板中挑取每种基因型的4-6个蠕虫,并测量它们对食物的逆转速率。在至少三个不同的测试日重复该测定,并且在每个测试日中,所有基因型均等同地表示
      4. 可逆测定基于公认的方案(例如,Tsalik和Hobert,2003)。每个蜗杆首先转移到板中没有食物的区域中,以便从其身体释放细菌残余物。一旦没有可见的食物痕迹,蠕虫被放置在6厘米的非种子NGM试验板上。在测定之前,将试验板放置1小时,其盖子关闭以干燥其表面。在将蠕虫转移到测试板后1分钟,在3分钟的持续时间内计数反转事件的数目。只有当蜗杆逆转时,它包括至少两个连续的头部弯曲,才计算反转。
        注意:我们在光刺激结束后2小时才观察到效果。

      数据分析

      每个实验在至少3个独立日进行,每天具有相似的重复次数。通过将反转事件的数量除以3分钟计算平均反转频率(每分钟反转的数量)。在应用2×2设计的实验中(例如,没有刺激对刺激和野生型蠕虫对机械感觉突变体),使用2×2方差分析来确定是否存在显着的相互作用之间的两个类别。然后使用Bonferroni校正的事后(post hoc)t检验在每个类别内进行比较。或者,在每个实验日没有任何刺激的情况下,通过平均逆转速率归一化逆转速率值,并且使用t检验来比较实验条件(例如,野生型型与机械感觉突变体)。原始数据的示例可以在 http://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.1002348#pbio.1002348.s001 ,这是对我们原始研究论文的补充( Rabinowitch 。,2016)。

      笔记

      1. C。 elegans 行为随其环境条件而变化很大。我们注意到,倒车率可能每天都有很大的变化。影响逆转速率的关键参数是测试板的干燥度。还有机械感觉突变体和野生型蠕虫之间逆转的差异变异性。这里一个关键因素是温度。为了克服这种变化并抵消其影响,每个实验在几个测试日分布。

      食谱

      1. 大肠杆菌菌株OP50
        1. 准备一瓶500毫升LB(根据产品配方)。
        2. 用单菌落OP50接种LB。
        3. 松开盖子,放在37℃的培养箱中
        4. 在不摇动的情况下生长过夜,第二天在4℃冰箱中生长
      2. 线虫生长培养基(NGM)(4L)
        1. 混合好:72g Bacto琼脂; 10g细菌蛋白胨和12g NaCl在4L玻璃烧瓶中
        2. 加入3.8L H 2 O。将搅拌棒放在烧瓶和高压釜中60分钟
        3. 紧接着高压釜加入:4ml 1M MgSO 4,100ml 1M磷酸钾缓冲液,pH 6.0和4ml 5mg/ml胆固醇。
        4. 将烧瓶置于55℃并搅拌
        5. 培养基冷却后,加入4ml 1M CaCl 2
        6. 同时仍然温暖在每个6厘米板10毫升。

      致谢

      支持编写协议由FHCRC新开发授权和NIH授予NINDS R01NS085214提供。目前的方案改编自以前的工作(Nagel等人,2005)

      参考文献

      1. Guo,ZV,Hart,AC和Ramanathan,S。(2009)。  的神经回路的光学探询 6(12):891-896。
      2. Kocabas,A.,Shen,CH,Guo,ZV and Ramanathan,S.(2012)。  控制在秀丽隐杆线虫中的中间神经元活性以诱发趋化行为。 Nature 490(7419):273-277。 />
      3. Leifer,AM,Fang-Yen,C.,Gershow,M.,Alkema,MJ和Samuel,AD(2011)。  自由移动秀丽隐杆线虫中的神经活动的光遗传学操作。 :147-152。
      4. Lindsay,TH,Thiele,TR and Lockery,SR(2011)。  对线虫的线虫的中枢神经系统中的突触传递的光遗传学分析。 Nat Commun 2:
      5. Nagel,G.,Brauner,M.,Liewald,JF,Adeishvili,N.,Bamberg,E.and Gottschalk,A。(2005)。  Caenorhabditis elegans的可兴奋细胞中的channelrhodopsin-2的光激活触发快速的行为反应。 > Curr Biol 15(24):2279-2284
      6. Rabinowitch,I.,Laurent,P.,Zhao,B.,Walker,D.,Beets,I.,Schoofs,L.,Bai,J.,Schafer,WR和Treinin, a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26745270"target ="_ blank">神经肽驱动的交感神经损伤后的跨模态可塑性elegans 。 PLoS Biol 14(1):e1002348。
      7. Stirman,JN,Crane,MM,Husson,SJ,Wabnig,S.,Schultheis,C.,Gottschalk,A。和Lu,H。(2011)。  自由表现秀丽隐杆线虫中的神经元和肌肉的实时多模态光学控制。 Nat Methods 8(2):153-158。
      8. Tsalik,EL和Hobert,O。(2003)。  在Caenorhabditis elegans中控制运动行为的神经元的功能映射。 J Neurobiol 56:178-197。
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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。

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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rabinowitch, I., Treinin, M. and Bai, J. (2016). Artificial Optogenetic TRN Stimulation of C. elegans. Bio-protocol 6(20): e1966. DOI: 10.21769/BioProtoc.1966; Full Text
  2. Rabinowitch, I., Laurent, P., Zhao, B., Walker, D., Beets, I., Schoofs, L., Bai, J., Schafer, W. R. and Treinin, M. (2016). Neuropeptide-driven cross-modal plasticity following sensory loss in Caenorhabditis elegans. PLoS Biol 14(1): e1002348.




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