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Fluorescein Transport Assay to Assess Bulk Flow of Molecules Through the Hypocotyl in Arabidopsis thaliana
荧光素转运测定法评估通过拟南芥下胚轴的分子总体流动   

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eLIFE
Jul 2017

Abstract

The bulk transport of molecules through plant tissues underpins growth and development. The stem acts as a conduit between the upper and low domains of the plant, facilitating transport of solutes and water from the roots to the shoot system, and sugar plus other elaborated metabolites towards the non-photosynthetic organs. In order to perform this function efficiently, the stem needs to be optimized for transport. This is achieved through the formation of vasculature that connects the whole plant but also through connectivity signatures that reduce path length distributions outside the vascular system. This protocol was devised to characterize how cell connectivity affects the bulk flow of molecules traversing the stem. This is achieved by exposing young seedlings to fluorescein, for which no specific transporter is assumed to be present in A. thaliana, and assessing the relative concentration of this fluorescent compound in individual cells of the embryonic stem (hypocotyl) using confocal microscopy and quantitative 3D image analysis after a given exposure time.

Keywords: Connectomics (连接组学), Tissue architecture (组织结构), Cellular networks (细胞网络), Bulk flow characterization (总体流动表征)

Background

The link between structure and function has always fascinated biologists, from the design spaces of organs (Eldredge, 1989) to the convergence or divergence of evolutionary paths (Morris, 2003). At a smaller scale, cells are also organized in a robust and tightly controlled manner, intimately related to the functions the tissue performs (Jackson et al., 2017a). The collection of cellular physical interactions that makes up a specific tissue can also be regarded as a network, a cellular connectome. This connectome is especially interesting in plants as shared cell walls impede cellular movement, thus the network dynamics depend only on cell death and replication.

We hypothesize that tissue architecture and thus cell connectomes are relevant to physiological features and organ function. This way, network metrics and quantitative networks analysis can be used to make predictions and gain understanding of biological systems (Duran-Nebreda and Bassel, 2017; Jackson et al., 2017b).

In the current example (Jackson et al., 2017a), we investigated the topological properties of different cell types in the embryonic stem by digitally capturing global cellular interactions using confocal microscopy, revealing systematic arrangements of reduced path length in the atrichoblast (non-hair forming) cell files. To address a possible functional link between the preferential movement of small molecules and this path length distribution, we developed a fluorescein transport assay. This involves exposing the embryos to fluorescein in a non-saturating manner and quantifying cell-type specific fluorescence following cell segmentation. Similar assays using fluorescein either with specific (Konishi et al., 2002; De Bruyn et al., 2011) or non-specific interactions (Wang and Fisher, 1994; Tichauer et al., 2015) exist, some using caged variants that allow for more control in activation and release (Kobayashi et al., 2007; Christensen et al., 2009). However, these did not provide a connection to global cellular connectivity and thus producing a general quantitative framework for structure-function relationships in tissue architecture and bulk transport processes.

Materials and Reagents

  1. 94 x 16 mm sterile Petri dishes (Greiner Bio One International, catalog number: 633181 )
  2. Barky Ultipette capillary tips (Barky Instruments International, catalog number: CP-100 )
  3. Aluminum foil or opaque container
  4. Cellview cell culture dish, 35 x 10 mm glass bottom (Greiner Bio One International, catalog number: 627861 )
  5. 1.5 ml Eppendorf tube
  6. Arabidopsis thaliana seeds
  7. Sterile distilled water (type I water pH 7.0)
  8. Bleach (Domestos)
  9. Ethanol
  10. Propidium iodide solution (Sigma-Aldrich, catalog number: P4864-10ML )
  11. Murashige and Skoog basal salt mixture with vitamins (Duchefa Biochemie, catalog number: M0222 )
  12. Agar-agar granular powder (Fisher Scientific, catalog number: A/1080/53 )
  13. Fluorescein (Alfa Aesar, catalog number: L13251 )
  14. Potassium hydroxide (Fisher Scientific, catalog number: P/5640/53 )
  15. ½ Murashige and Skoog medium (see Recipes)
  16. Fluorescein plates (see Recipes)

Equipment

  1. Tissue culture hood (Azbil Telstar, model: AH-100 )
  2. 20 μl pipette (Gilson, model: PIPETMAN P20L )
  3. Small forceps (IDEAL-TEK, model: 4.SA.0 )
  4. Inverted confocal microscope (ZEISS, model: LSM 710 )
  5. Dissection microscope (Leica Microsystems stereo microscope, Leica Microsystems, model: Leica S6 E )
  6. 1,000 μl pipette (Gilson, model: PIPETMAN P1000L )
  7. Heated bath or microwave
  8. Water purification system (ELGA LabWater, model: Option-R 7 )
  9. 500 ml Pyrex bottles (DWK Life Sciences, DURAN, catalog number: 21 801 44 )
  10. Autoclave (Dixons, catalog number: ST 2228 )
  11. pH-meter (Hanna Instruments, model: pH 210 )
  12. Growth cabinet or room (16 h light photoperiod with light intensity at 150-175 mmol m2 sec-1 at 23 °C and 8 h dark at 18 °C)

Software

  1. ImageJ (Schindelin et al., 2012) with the Bio-formats plug-in
  2. MorphoGraphX (Barbier de Reuille et al., 2015)
    Note: Uses the CUDA toolkit, software developers recommend NVidia graphics card and enough memory to handle the stacks being processed.

Procedure

  1. Germinating the seeds
    1. Prepare in advance ½ MS Petri dishes as described in the ‘Recipes’ section.
    2. Prepare a fresh 1/10 dilution of the commercial bleach with distilled water to sterilize the seeds.
    3. Place 60-100 seeds of each ecotype or species in a separate 1.5 ml Eppendorf tube and add 500 μl of the bleach solution to each tube.
    4. Incubate at RT for 5 min, mix by inverting the tube every minute.
    5. Move to the tissue culture hood.
    6. Sterilize the flexible pipette tips with ethanol.
    7. Pipette out the bleach and wash the seeds three times with 500 μl of sterile distilled water.
    8. Pick a string (10-30) of sterile seeds with a P20 pipette and the flexible pipette tips and put the seeds one by one and approximately 5 mm apart in a ½ MS plate.
    9. Cover the Petri dishes with aluminum foil or an opaque container and take them to the growth room or cabinet at 23 °C.
    10. Incubate in complete darkness for 4 days.
      Note: This causes the seedlings to be elongated and to contain very little chlorophyll, which reduces autofluorescence of the sample during imaging, yielding better signal.

  2. Fluorescein incubation treatment
    1. Prepare on the same day a batch of fluorescein-agar plates as described in the ‘Recipes’ section.
    2. Move the seedlings from the ½ MS plates to the fluorescein 0.8% agar Petri dishes using the small tweezers. Do not squeeze the seedlings as their walls are very thin at this point, lift them instead by putting the tweezer prongs under the cotyledons with minimal pressure. Place their root onto the agar surface such that the seedling does not fall onto its side. Use the dissection microscope to ease handling of the seedlings. Roots can be gently manipulated into the agar to ensure the seedling remains upright.
    3. Incubate the seedlings in the growth room for 2.5 h under the light source to maximize fluorescein uptake.

  3. Propidium iodide staining
    1. Create a 5 μg/ml propidium iodide solution in water and place it in as many 1.5 ml Eppendorf tubes as needed.
    2. Transfer the seedlings into these Eppendorf tubes (10-20 in each) using the same technique as before to avoid damaging them.
    3. Incubate for 15 min at RT, mix by gently inverting the tube.
    4. Move the seedlings from the Eppendorf tubes to imaging dishes (Cellview cell culture dishes) using a P1000 and a cut pipette tip. The dishes should contain as many non-overlapping samples as possible, usually between 3 and 5. The dissection microscope can be used at this stage to detect and remove damaged samples (cracks in the epidermis, snapped roots or squeezed sections).
    5. Wash once with 300 μl of sterile distilled water and remove as much water as possible by pippeting, in such a way that the seedlings still have a layer of liquid around them. When imaging, add sterile distilled water as needed if the samples dry out.

  4. Imaging
    The following list contains a typical list of settings to image these samples:
    1. 25x oil immersion objective.
    2. Zoom 1 (this can be changed to better suit the size of the ROI, although systematic warps to the images appear < 0.7 zoom).
    3. Frame size: 2,048 x 2,048. This can be adjusted to the proportions of the ROI.
    4. Bit depth: 16 bits.
    5. Pinhole slice thickness: 1.9 μm (0.47 AU).
    6. Slice interval (z direction): 0.7 μm.
    7. Scan speed: 9.
    8. Averaging: 4-8.
    9. Fluorescein excitation: 488 nm.
    10. Propidium iodide excitation: 535 nm.

Data analysis

The data obtained with this protocol should be processed using the same protocol described previously in Montenegro-Johnson et al. (2015) and Jackson et al. (2017a and 2017b). Namely, the propidium iodide channel is used to perform 3D segmentation of the cell boundaries as it stains cell walls, while the fluorescein signal within each cell is used to characterize bulk flow for each cell type in the hypocotyl. See Figure 1 for a typical example of the obtained confocal stacks before processing. The steps are as follows:

  1. Load the confocal stacks into Fiji using the Bio-formats plugin and export each channel to a TIFF image format stack.
  2. Load each TIFF stack onto MorphoGraphX.
  3. Apply a Gaussian blur (typically 0.3 μm smooth length in each direction) to the propidium iodide channel.
  4. Use the ITK watershed segmentation on the smoothed propidium iodide stack to find the cell boundaries. The threshold used varies depending on the staining and image acquisition settings.
  5. Edit the stacks by fusing oversegmeneted cells as needed.
  6. Create a mesh for the cells with ‘cube size: 2’ and no smooth passes as settings.
  7. Calculate fluorescein concentration using the heat map function with ‘volume’ and ‘internal signal’ settings.

The three ecotypes used in the original study presented different average fluorescent readings, possibly due to innate differences in permeability and/or bulk uptake rates. Some ecotypes also displayed far greater variability than others. For this reason, in order to be pooled together all samples need to be normalized by within-sample mean fluorescence. Then to be comparable between ecotypes all pooled data has to be normalized by ecotype-wide mean corrected fluorescence.


Figure 1. Typical results of imaging Arabidopsis hypocotyls after fluorescein exposure and propidium iodide staining. A. Single confocal stack of an Arabidopsis root, with root hairs visible. Two channels are shown, in grey scale propidium iodide, which stains cell walls, and in green fluorescein moving inside the living cells. B. 3D reconstruction of a hypocotyl from dozens of confocal stacks in MorphoGraphX. Transparency is used to show fluorescein signal accumulated inside the first layer of cells. C. A Transversal clip from the 3D reconstruction reveals a pattern of fluorescein concentration correlating with cell type spatial arrangements. This allows us to address which cells are involved in greater bulk flow through the epidermis.

Recipes

  1. ½ Murashige and Skoog medium (Murashige and Skoog, 1962)
    2.3 g/L of Murashige and Skoog salt mixture with vitamins
    0.8 g/L of agar-agar granulated powder
    Add 80% of the final volume of filter-purified water (type I water, > 18.2 MΩ-cm)
    Adjust pH with a 1 M KOH solution to 6.2
    Top to selected final volume with filter-purified water (type I water, > 18.2 MΩ-cm)
    Autoclave and pour into sterile Petri dishes (20 ml/dish) inside a tissue culture hood. Store poured Petri dishes at 4 °C before use (1 month shelf life)
  2. Fluorescein plates
    1. Prepare in advance a 0.8 g/L agar-agar granulated powder mixture (follow the previous recipe but without adding the Murashige and Skoog salt mixture) and store at RT
    2. Prepare a 50 mM fluorescein solution (1,000x stock in 1:1 ethanol:sterile distilled water) and store it avoiding direct light sources (2 months shelf life)
    3. Melt the agar gel with a heated bath, steamer or by microwaving the gel thoroughly
    4. Wait until the solution cools off, reaching 50-60 °C
    5. Add the 1,000x fluorescein stock solution and pour the mix into Petri dishes (15 ml/dish). This need not be sterile and can be poured outside a tissue culture hood

Acknowledgments

This work was supported by BBSRC grants BB/J017604/1, BB/L010232/1 and BB/N009754/1 to GWB, and by Leverhulme Trust Grant RPG-2016–049 to S.D.-N. and GWB. The authors declare no conflict of interest.

References

  1. Barbier de Reuille, P., Routier-Kierzkowska, A. L., Kierzkowski, D., Bassel, G. W., Schupbach, T., Tauriello, G., Bajpai, N., Strauss, S., Weber, A., Kiss, A., Burian, A., Hofhuis, H., Sapala, A., Lipowczan, M., Heimlicher, M. B., Robinson, S., Bayer, E. M., Basler, K., Koumoutsakos, P., Roeder, A. H., Aegerter-Wilmsen, T., Nakayama, N., Tsiantis, M., Hay, A., Kwiatkowska, D., Xenarios, I., Kuhlemeier, C. and Smith, R. S. (2015). MorphoGraphX: A platform for quantifying morphogenesis in 4D. Elife 4: 05864.
  2. Christensen, N. M., Faulkner, C. and Oparka, K. (2009). Evidence for unidirectional flow through plasmodesmata. Plant Physiol 150(1): 96-104.
  3. De Bruyn, T., Fattah, S., Stieger, B., Augustijns, P. and Annaert, P. (2011). Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3. J Pharm Sci 100(11): 5018-5030.
  4. Duran-Nebreda, S. and Bassel, G. W. (2017). Bridging scales in plant biology using network science. Trends Plant Sci 22(12): 1001-1003.
  5. Eldredge, N. (1989). Macroevolutionary Dynamics: Species, Niches and Adaptive Peaks. McGraw Hill.
  6. Jackson, M. D., Xu, H., Duran-Nebreda, S., Stamm, P. and Bassel, G. W. (2017a). Topological analysis of multicellular complexity in the plant hypocotyl. Elife 6: e26023.
  7. Jackson, M. D. B., Duran-Nebreda, S. and Bassel, G. W. (2017b). Network-based approaches to quantify multicellular development. J R Soc Interface 14(135).
  8. Kobayashi, T., Urano, Y., Kamiya, M., Ueno, T., Kojima, H. and Nagano, T. (2007). Highly activatable and rapidly releasable caged fluorescein derivatives. J Am Chem Soc 129(21): 6696-6697.
  9. Konishi, Y., Hagiwara, K. and Shimizu, M. (2002). Transepithelial transport of fluorescein in Caco-2 cell monolayers and use of such transport in in vitro evaluation of phenolic acid availability. Biosci Biotechnol Biochem 66(11): 2449-2457.
  10. Montenegro-Johnson, T. D., Stamm, P., Strauss, S., Topham, A. T., Tsagris, M., Wood, A. T., Smith, R. S. and Bassel, G. W. (2015). Digital single-cell analysis of plant organ development using 3DCellAtlas. Plant Cell 27(4): 1018-1033.
  11. Morris, S. C. (2003). Life’s solution: inevitable humans in a lonely universe. Cambridge University Press.
  12. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.
  13. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  14. Tichauer, K. M., Guthrie, M., Hones, L., Sinha, L., St Lawrence, K. and Kang-Mieler, J. J. (2015). Quantitative retinal blood flow mapping from fluorescein videoangiography using tracer kinetic modeling. Opt Lett 40(10): 2169-2172.
  15. Wang, N. and Fisher, D. B. (1994). The use of fluorescent tracers to characterize the post-phloem transport pathway in maternal tissues of developing wheat grains. Plant Physiol 104(1): 17-27.

简介

分子通过植物组织的大量运输支撑了生长和发育。茎部充当植物上部和低部位之间的导管,促进溶质和水从根部向茎部系统的运输,糖和其他精细代谢物向非光合器官转运。为了有效地执行此功能,杆需要针对运输进行优化。这通过形成连接整个植物的脉管系统来实现,但也通过连接特征来减少血管系统外的路径长度分布。该协议被设计为描述细胞连接如何影响穿过茎的分子的大量流动。这是通过将幼苗暴露于荧光素而实现的,其中假定没有特定的转运蛋白存在于A中。在给定的暴露时间后,使用共焦显微镜和定量3D图像分析评估该荧光化合物在胚胎干(下胚轴)的单个细胞中的相对浓度。

【背景】结构和功能之间的联系一直着迷于生物学家,从器官的设计空间(Eldredge,1989)到进化路径的趋同或分歧(Morris,2003)。在较小规模的情况下,细胞也以稳健且严格控制的方式组织,与组织执行的功能密切相关(Jackson等人,2017a)。构成特定组织的细胞物理相互作用的集合也可被视为网络,即细胞连接体。这种连接体在植物中特别有趣,因为共享细胞壁阻碍细胞运动,因此网络动力学仅取决于细胞死亡和复制。

我们假设组织结构和细胞连接体与生理特征和器官功能有关。这样,网络指标和定量网络分析可用于预测并获得生物系统的理解(Duran-Nebreda和Bassel,2017; Jackson等人,2017b)。

在当前的例子中(Jackson等人,2017a),我们通过使用共聚焦显微镜以数字方式捕获全局细胞相互作用来研究胚胎干中不同细胞类型的拓扑性质,揭示了缩短的路径长度的系统布置在atrichoblast(非毛发形成)细胞文件中。为了解决小分子优先移动和该路径长度分布之间可能的功能联系,我们开发了荧光素转运测定法。这涉及以非饱和方式将胚胎暴露于荧光素并在细胞分割后量化细胞类型特异性荧光。类似的使用荧光素与特异性(Konishi等人,2002; De Bruyn等人,2011)或非特异性相互作用(Wang和Fisher,1994; Tichauer其中一些使用允许更多控制激活和释放的笼式变体(Kobayashi et al。,2007; Christensen et al。 ,2009)。然而,这些并没有提供与全球细胞连接的连接,从而为组织结构和散装运输过程中的结构 - 功能关系提供了一个通用的量化框架。

关键字:连接组学, 组织结构, 细胞网络, 总体流动表征

材料和试剂


  1. 94×16毫米无菌培养皿(Greiner Bio One International,目录号:633181)
  2. Barky Ultipette毛细管吸头(Barky Instruments International,目录号:CP-100)
  3. 铝箔或不透明的容器
  4. Cellview细胞培养皿,35×10mm玻璃底(Greiner Bio One International,目录号:627861)
  5. 1.5毫升Eppendorf管
  6. 拟南芥种子
  7. 无菌蒸馏水(I型水pH 7.0)
  8. 漂白剂(Domestos)
  9. 乙醇
  10. 碘化丙锭溶液(Sigma-Aldrich,目录号:P4864-10ML)
  11. 含有维生素的Murashige和Skoog基础盐混合物(Duchefa Biochemie,目录号:M0222)
  12. 琼脂颗粒状粉末(Fisher Scientific,目录号:A / 1080/53)
  13. 荧光素(Alfa Aesar,目录号:L13251)
  14. 氢氧化钾(Fisher Scientific,目录号:P / 5640/53)
  15. ½Murashige和Skoog中(见食谱)
  16. 荧光素板(见食谱)

设备

  1. 组织培养罩(阿兹比勒Telstar,型号:AH-100)
  2. 20微升移液器(吉尔森,型号:PIPETMAN P20L)
  3. 小镊子(IDEAL-TEK,型号:4.SA.0)
  4. 倒置共焦显微镜(ZEISS,型号:LSM 710)
  5. 解剖显微镜(徕卡显微系统体视显微镜,徕卡显微系统,型号:徕卡S6 E)
  6. 1,000微升移液器(吉尔森,型号:PIPETMAN P1000L)
  7. 加热浴缸或微波炉
  8. 水净化系统(ELGA LabWater,型号:Option-R 7)
  9. 500毫升Pyrex瓶(DWK生命科学公司,DURAN,目录号:21 801 44)
  10. 高压灭菌器(Dixons,目录号:ST 2228)
  11. pH计(Hanna Instruments,型号:pH210)
  12. 生长室或房间(光照强度为150-175毫摩尔m 2 / sec-1在23℃和18小时黑暗下18小时的光照时间为16小时)

软件

  1. ImageJ(Schindelin等人,2012)与Bio-formats插件
  2. MorphoGraphX(Barbier de Reuille et al。,2015)
    注:使用CUDA工具包,软件开发人员推荐NVidia图形卡和足够的内存来处理正在处理的堆栈。

程序

  1. 发芽的种子
    1. 按照“食谱”部分中的描述预先准备好½个培养皿。

    2. 用蒸馏水准备1/10稀释的商用漂白剂以消毒种子。
    3. 将每种生态型或物种的60-100个种子放入单独的1.5ml Eppendorf管中,并将500μl漂白剂溶液添加到每个管中。

    4. 在室温下孵育5分钟,每分钟颠倒一次管混合。
    5. 移动到组织培养罩。

    6. 用乙醇消毒软管吸头。

    7. 移取漂白剂,用500μl无菌蒸馏水清洗种子三次
    8. 用P20移液管和柔性移液管吸头挑选一串(10-30)无菌种子,并将种子逐个放入一个½MS板中,间隔约5 mm。

    9. 用铝箔或不透明容器覆盖培养皿,并将它们放在23°C的培养室或橱柜中。

    10. 在完全黑暗中孵育4天 注意:这会导致幼苗拉长并且含有很少的叶绿素,从而减少成像过程中样品的自发荧光,产生更好的信号。

  2. 荧光素孵育处理

    1. 在同一天准备一批荧光琼脂平板,如“食谱”部分所述。
    2. 使用小镊子将½MS板上的幼苗移至荧光素0.8%琼脂培养皿中。不要挤压幼苗,因为此时幼苗的幼苗壁很薄,取而代之的是用最小的压力将镊子尖放在子叶下面。将它们的根放在琼脂表面上,使幼苗不会落到其侧面。使用解剖显微镜来缓解幼苗的处理。
      。根可以轻轻地操纵进入琼脂,以确保幼苗保持直立。

    3. 在光源下培育2.5小时的幼苗,以最大限度地提高荧光素摄取量。

  3. 碘化丙啶染色
    1. 在水中制备5μg/ ml碘化丙啶溶液,根据需要放入1.5 ml Eppendorf管中。
    2. 使用与以前相同的技术将苗移入这些Eppendorf管(每个10-20),以避免损坏它们。

    3. 在RT下孵育15分钟,通过轻轻翻转管进行混合。
    4. 使用P1000和切口移液枪头将幼苗从Eppendorf管移至成像皿(Cellview细胞培养皿)。盘子应该包含尽可能多的不重叠的样品,通常在3到5之间。解剖显微镜可以在这个阶段用来检测和去除受损的样品(表皮中的裂缝,咬合的根部或挤压部分)。 >
    5. 用300μl无菌蒸馏水洗一次,并尽可能多地用水去除,这样幼苗周围仍有一层液体。成像时,如果样品干燥,则根据需要添加无菌蒸馏水。

  4. 成像
    以下列表包含用于对这些样本进行成像的典型设置列表:
    1. 25倍油浸物镜。
    2. 缩放1(尽管图像的系统变形显示<0.7变焦),但可以更改为更适合ROI的大小。
    3. 帧大小:2,048 x 2,048。这可以根据ROI的比例进行调整。
    4. 位深度:16位。
    5. 针孔切片厚度:1.9μm(0.47 AU)。
    6. 切片间隔(z方向):0.7μm。
    7. 扫描速度:9.
    8. 平均值:4-8。
    9. 荧光素激发:488nm。
    10. 碘化丙锭激发:535纳米。

数据分析

使用这个协议获得的数据应该使用前面在Montenegro-Johnson et al。(2015)和Jackson 等人描述的相同协议进行处理。 (2017a和2017b)。即,碘化丙啶通道用于在细胞壁污染细胞壁时进行细胞边界的3D分割,而每个细胞内的荧光素信号用于表征下胚轴中每种细胞类型的体积流量。处理前获得的共焦堆栈的典型示例见图1。步骤如下:

  1. 使用生物格式插件将共焦堆栈加载到斐济,并将每个通道导出为TIFF图像格式堆栈。
  2. 将每个TIFF堆叠加载到MorphoGraphX上。
  3. 应用高斯模糊(每个方向通常为0.3μm光滑长度)到碘化丙啶通道。
  4. 在平滑的碘化丙锭堆栈上使用ITK分水岭来分割单元边界。使用的阈值取决于染色和图像采集设置。

  5. 根据需要融合违规单元格来编辑堆栈

  6. 为“多维数据集大小:2”的单元格创建网格,并且没有平滑的传递作为设置
  7. 使用具有“音量”和“内部信号”设置的热图功能计算荧光素浓度。

原始研究中使用的三种生态型呈现不同的平均荧光读数,可能是由于渗透性和/或体积摄入率的天然差异。一些生态型也显示出比其他生态型更大的变异性。为此,为了将所有样品汇集在一起,需要通过样品内平均荧光进行标准化。然后为了在生态型之间进行比较,所有汇总的数据必须通过生态型广义平均修正荧光进行标准化。


图1.荧光素暴露和碘化丙啶染色后成像的拟南芥下胚轴的典型结果A.拟南芥根与根的单共焦堆叠,头发可见。图中显示了两个通道,分别是灰色碘化丙啶碘化物,它染色细胞壁,绿色荧光素在活细胞内移动。 B. MorphoGraphX中几十个共焦堆栈的下胚轴3D重建。透明度用于显示在第一层细胞内累积的荧光素信号。 C.来自3D重建的横向剪辑揭示了与细胞类型空间排列相关的荧光素浓度模式。这允许我们解决哪些细胞参与通过表皮更大的体积流量。

食谱

  1. ½Murashige和Skoog中(Murashige和Skoog,1962年)
    2.3克/升Murashige和Skoog盐混合物与维生素
    0.8克/升的琼脂造粒粉
    添加过滤净化水(I型水,> 18.2MΩ-cm)的最终体积的80%
    用1M KOH溶液调节pH至6.2
    使用过滤纯化水(I型水,> 18.2MΩ-cm)选择最终体积。
    高压灭菌器并倒入组织培养罩内的无菌培养皿(20ml /皿)。
    在使用前储存4°C的培养皿(保质期为1个月)
  2. 荧光素板
    1. 事先准备好0.8克/升琼脂 - 琼脂造粒粉末混合物(按照以前的配方,但不添加Murashige和Skoog盐混合物)并在RT储存。
    2. 准备50 mM荧光素溶液(1:1乙醇:无菌蒸馏水中的1,000x储备液)并储存,避免直接光源(储存期为2个月)
    3. 融化琼脂凝胶,加热浴缸,蒸锅或通过彻底微波凝胶
    4. 等待溶液冷却,达到50-60°C
    5. 加入1,000x荧光素原液并将混合物倒入培养皿(15ml /皿)中。这不必是无菌的,可以倒在组织培养罩
      外面

致谢

这项工作得到了BBSRC的支持,BB / J017604 / 1,BB / L010232 / 1和BB / N009754 / 1授予GWB,Leverhulme Trust Grant RPG-2016-049授予S.D.-N.和GWB。作者宣称没有利益冲突。

参考

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Copyright Duran-Nebreda and Bassel. 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. Duran-Nebreda, S. and Bassel, G. W. (2018). Fluorescein Transport Assay to Assess Bulk Flow of Molecules Through the Hypocotyl in Arabidopsis thaliana. Bio-protocol 8(7): e2791. DOI: 10.21769/BioProtoc.2791.
  2. Jackson, M. D., Xu, H., Duran-Nebreda, S., Stamm, P. and Bassel, G. W. (2017a). Topological analysis of multicellular complexity in the plant hypocotyl. Elife 6: e26023.
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