参见作者原研究论文

本实验方案简略版
May 2019

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Method for Measuring Mucociliary Clearance and Cilia-generated Flow in Mice by ex vivo Imaging
小鼠中黏膜纤毛清除和纤毛产生流动的离体成像检测方法   

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Abstract

Ex vivo biophysical measurements provide valuable insights into understanding both physiological and pathogenic processes. One critical physiological mechanism that is regulated by these biophysical properties is cilia-generated flow that mediates mucociliary clearance, which is known to provide protection against foreign particles and pathogens in the upper airway. To measure ciliary clearance, several techniques have been implemented, including the use of radiolabeled particles and imaging with single-photon emission computerized tomography (SPECT) methods. Although non-invasive, these tests require the use of specialized equipment, limiting widespread use. Here we describe a method of ex vivo imaging of cilia-generated flow, adapted from previously reported methods, to make it more accessible and higher throughput for researchers. We excise trachea from mice quickly after euthanasia, cut it longitudinally and place it in an inhouse made slide. We apply fluorescent particles to measure particle movement under a fluorescent microscope, followed by analysis with ImageJ, allowing calculation of fluid flow generated by cilia under different conditions. This method enables ex vivo measurements in tissue with minimal investment or special equipment, giving opportunity to investigate and discover important biophysical properties associated with ciliary movement of the trachea in physiology and disease.

Keywords: Cilia (纤毛), Biophysics (生物物理), Mucociliary clearance (黏膜纤毛清除), Lung (肺), Ex-vivo imaging (离体成像)

Background

The composition of diverse biophysical properties, each aiding a specified function, make up the human body. A key example of this is the generation of flow in many different organ systems, including the central nervous system (Olstad et al., 2019), reproductive tracts (Afzelius et al., 1978; Yuan et al., 2019) and the pulmonary system (Satir and Sleigh, 1990), made by specialized ciliated cells. Understanding how the environment or external stimuli affect these processes can give us insights into disease. In our recent study (Kudo et al., 2019), we explored how humidity affects influenza infections in a mouse model. By using this method of ex-vivo measurement of cilia-generated flow in mice, we were able to visually measure how ambient humidity altered the rate of virus clearance (Figures 1 and 2). Previous studies have used radiolabeled particles to measure mucociliary clearance using a noninvasive method (Hua et al., 2010; Bustamante-Marin and Ostrowski, 2017). However, this requires the use of radioactive materials and SPECT or other machines to measure radioactivity that may not be readily available. Measurements like this can be made more accessible using ex vivo imaging of particles in tissue (Nance et al., 2012; Francis and Lo, 2013; Mastorakos et al., 2015 and 2016). Here, we describe a detailed method of measuring the flow generated by cilia in upper airway tissue, adapted from Francis and Lo (2013). Our method is easy to perform and allows for measurement of large numbers of mice at a relatively low cost. This protocol can also be performed in a short time, allowing for the preservation of biological differences and measurement in a large number of animals. Finally, it can be adopted for other organ systems or tissues, measuring various biophysical properties, as shown by Nance et al. (2012), in identifying what criteria drug loaded particles must meet to navigate through brain parenchyma. Altogether, this protocol will provide researchers with an easy to follow, step-by-step methodology to measure properties of biological tissues ex vivo (Figure 3).

Materials and Reagents

  1. Superfrost Plus Gold Slides (Thermo Fisher Scientific, catalog number: FT4981GLPLUS ). Brand is not critical
  2. Square Cover Slips (Thermo Fisher Scientific, catalog number: 18X18-1 ). Brand is not critical
  3. Electrical tape (3M Safety, Scotch, Purchase in Amazon). Brand is not critical
  4. FluoSpheres carboxylate, 0.2 μm crimson, 625/645 (Life Technologies, catalog number: F8806 ) Alternate sizes/chemistry can be used depending on experimental design, refer to background above
  5. 6-well plate (Fisher Scientific, Corning). Brand is not critical, does not have to be tissue culture quality
  6. Mice
  7. PBS (Millipore Sigma, catalog number: D8537 ). Brand is not critical
  8. Super glue (Loctite, Purchase in Amazon). Brand is not critical
  9. 1:1,000 volume/volume of FluoSpheres carboxylate in PBS (see Recipes)

Equipment

  1. Dissection scissors (Fine Science Tools, catalog number: 14061-10 ) Brand is not critical
  2. Electric Razor (Purchase from Amazon) Brand is not critical
  3. Hair removal product, Nair (Purchase from Amazon) Brand is not critical
  4. Forceps (Fine Science Tools) Brand is not critical
  5. Micro dissection scissors (Fine Science Tools) Brand is not critical
  6. Scalpel (Purchase from Amazon) Optional and brand is not critical
  7. Dissection microscope (Laxco, LMS-Z200) Optional and brand is not critical
  8. Bruker Opterra Swept Field Microscope (Bruker) Brand is not critical, microscope must be equipped with high-speed fluorescent camera (Photometrics Evolve Delta EMCCD camera)

Software

  1. Obtaining images Prairie View 5.4 (Bruker) Brand is not critical, any software associated with microscope will work
  2. Analysis Software ImageJ (Directionality, Mtrack2, Manual Tracking, NIH, https://imagej.nih.gov/ij/) Other software can be used for analysis

Procedure

  1. Prepare slides
    1. Make a stack of electrical tape to a thickness of ~1.5 mm (7 pieces of tape) (Figures 1A-1B).
      Note: The thickness of the tape here can vary depending on the thickness of tissue you are interested in working with, with the thickness corresponding to the tissue touching both the bottom of the slide and the coverslip without distorting the tissue significantly. However, it is important to keep the thickness consistent between samples, as thickness can affect your imaging later on.
    2. Cut a small section using a scalpel to allow for housing of the trachea (Figure 1C).
    3. Put the tape on a slide to make a slide with a housing chamber usable with most microscopes (Figure 1D).
      Note: Slides can be prepared in large numbers easily before experiment. Do this to speed up throughput of experiments.


      Figure 1. Preparation of chamber slides for measurement of mucociliary clearance. A-B. Electrical tape was stacked on top of each other to make a stack equivalent to ~1.5 mm (white arrow). C. Sections were cut in the center of the tape to create a chamber for tissue. D. Tape was attached to slide for imaging. E. Example of Tracheal tissue that was cut longitudinally and placed in the chamber with cover slip.

  2. Dissection of trachea (Time sensitive)
    The following procedure, till the image acquisition should be performed uninterrupted for each mouse (rather than extracting all tracheae for the experiments) and quickly for best results.
    1. Euthanize mice in accordance with local institutional animal care and use committee (IACUC) policies and protocol.
    2. Immobilize mouse using a dissection board or by taping mouse onto a hard surface. Immediately expose neck skin using a hair removal product of your choice (Nair, Electric Razor), make a ~3 cm incision from the chest to nose, and move salivary glands aside to expose trachea of mice.
    3. Clean connective tissue associated with trachea and make transverse cut on the superior end of the trachea. Keep trachea taut by holding this side with forceps.
    4. Cut inferior end of trachea.
    5. Immediately transfer to 6 well plate with ~1 ml PBS.
    6. Cut longitudinally through trachea using the micro dissection scissors.
    7. Immediately continue onto next step.

  3. Mounting trachea
    1. Prepare a solution of 1:1,000 of FluoSpheres carboxylate in PBS.
      Note: Prepare large batches for use of your entire experiment. Vortex initial solution well, and vortex the mix well between each use. 
    2. Place trachea from Procedure B into slide from Procedure A with the lumen side facing up.
      Note: Having the tape height match the tissue thickness is critical, as this allows the coverslip to prevent the trachea from refolding during imaging. 
    3. Place ~250 μl of 1:1,000 FluoSphere solution in the chamber, place a few drops of super glue on edges of tape and coverslip (Figure 1E).
      Note: Take precaution not to put excess liquid in chamber and to not put excess glue on tape. Aim for having enough liquid to submerge sample, or fully covering the center, without it spilling over. Mixing of the liquid submerging the sample with the super glue will result in unreliable results. Optimize with practice slides to get exact volume needed for each setup.
    4. Immediately continue onto next step.

  4. Imaging
    1. Microscope should be turned on and set into the correct position. Use a slide with particle solution but no organ as a positive control (refer to Video 1).
      Note: Imaging was performed under room temperature. However, experiments can be performed under different temperatures if your microscope setup allows for different temperature settings.

      Video 1. Example of particles moving in free solution in a brownian motion

    2. Place slide into position.
    3. *Important* Change focus until you find a plane where particles seem to be static. This is the plane of the tissue, where particles are attached/engulfed to the mucus/cells on the surface. From there move focus up a set amount for each image. This is important because the measurement of the flow generated by cilia will change as a function of the distance away from the surface of the cilia. Thus, to generate data that can be compared between samples, this distance must be kept constant (refer to Video 2). In our experiments, we used a distance of ~1 μm distance from the surface of the cilia.

      Video 2. Example of particles moving with directionality due to cilia generated flow

    4. Acquire image at speeds at or above 100 frames per second (we acquired at 100 fps for 30 s). Images were taken with microscope settings; Image Size: 512 μm x 512 μm, FOV: 204.2 μm x 204.2 μm, and Pixel Size 0.399 μm x 0.399 μm.
      Note: The two imaging lasers in our experiments were 488 nm and 561 nm, with the emission filters optimized for GFP and RFP. The two cameras on the microscope are 512 μm x 512 μm Photometrics Evolve Delta EMCCD cameras. The Laser is scanned with either a slit or an array of 32 pinholes, which is designed to increase scan speed, which is 67 fps max speed at full frame, with smaller ROIs allowing for much faster speeds.

    5. It is critical that these images are taken as quickly as possible; in our hands, all images were taken within 5 min of dissection, and saw decreased viability of tissue after 10 min.
      Note: The key component to this step is feeling comfortable with the procedure and decreasing the time between dissection and imaging. Practice to get a good flow going, or work in pairs to have one person dissect while another sets up the microscope in order to set up an optimal imaging condition. As a positive control of cilia movement regulation, mice housed in higher ambient humidity (~50%) will show higher cilia particle velocity (Kudo et al., 2019). As a negative control, mouse housed in lower ambient humidity can be used, or trachea that has be excised for a prolonged time period (> 10 min) can be used.

Data analysis

  1. Load data into FIJI/ImageJ using File > Import > Image sequence.
  2. Analyze particle velocity using Plugins > Tracking > MTrack2 (https://imagej.net/MTrack2).
    Note: Use imageJ settings Image > Adjust > Brightness/Contrast to ensure a high signal to noise ratio. This will allow for the MTrack2 program to identify the particles better.
  3. Measure single particle trajectory by using Plugins > Tracking > Manual Tracking (https://imagej.nih.gov/ij/plugins/track/track.html) (Figure 2).
    Note: This function will allow for coordinates of a single particle to be recorded. This can be then graphed using a graphing software to show the entire trajectory of a particle over a given time.
  4. Calculate directionality of flow using Analyze > Directionality (Figure 3).
  5. Export video (Videos 1 and 2) versions of particle flow using File > Save As > AVI.


    Figure 2. Cilia generated flow and directionality analysis of Videos 1 and 2. A. Quantification of cilia generated flow in Videos 1 and 2 using Data analysis section of the protocol. B-C. Quantification of frequency of directionality of Videos 1 and 2.


    Figure 3. Schematic of principal steps in cilia generated flow measurement

Recipes

  1. 1:1,000 volume/volume of FluoSpheres carboxylate in PBS
    Solution should be stored in the dark
    We made fresh solution each day and equilibrated to room temperature before use

Acknowledgments

We thank Melissa Linehan for technical and logistical assistance. We thank Al Mennone with help setting up the microscope for these experiments and Eriko Kudo for experiments that were performed that led to the original research paper. Graphical illustrations were made with Biorender.com. This work was supported in part by the Howard Hughes Medical Institute (A.I.), a gift from the Condair Group and National Institutes of Health Grants T32GM007205 (Medical Scientist Training Program training grant to E.S.). The original research paper for this protocol can be found at Kudo et al. (2019).

Competing interests

The authors have no financial and non-financial competing interests regarding the protocols described in this manuscript.

Ethics

No human subjects were used in this study. All animal work was approved under Yale IACUC protocol 2018-10365.

References

  1. Afzelius, B. A., Camner, P. and Mossberg, B. (1978). On the function of cilia in the female reproductive tract. Fertil Steril 29(1): 72-74.
  2. Bustamante-Marin, X. M. and Ostrowski, L. E. (2017). Cilia and mucociliary clearance. Cold Spring Harb Perspect Biol 9(4).
  3. Francis, R. and Lo, C. (2013). Ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia. J Vis Exp(78).
  4. Hua, X., Zeman, K. L., Zhou, B., Hua, Q., Senior, B. A., Tilley, S. L. and Bennett, W. D. (2010). Noninvasive real-time measurement of nasal mucociliary clearance in mice by pinhole gamma scintigraphy. J Appl Physiol (1985) 108(1): 189-196.
  5. Kudo, E., Song, E., Yockey, L. J., Rakib, T., Wong, P. W., Homer, R. J. and Iwasaki, A. (2019). Low ambient humidity impairs barrier function and innate resistance against influenza infection. Proc Natl Acad Sci U S A 116(22): 10905-10910.
  6. Mastorakos, P., da Silva, A. L., Chisholm, J., Song, E., Choi, W. K., Boyle, M. P., Morales, M. M., Hanes, J. and Suk, J. S. (2015). Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy. Proc Natl Acad Sci U S A 112(28): 8720-8725.
  7. Mastorakos, P., Song, E., Zhang, C., Berry, S., Park, H. W., Kim, Y. E., Park, J. S., Lee, S., Suk, J. S. and Hanes, J. (2016). Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain. Small 12(5): 678-685.
  8. Nance, E. A., Woodworth, G. F., Sailor, K. A., Shih, T. Y., Xu, Q., Swaminathan, G., Xiang, D., Eberhart, C. and Hanes, J. (2012). A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue. Sci Transl Med 4(149): 149ra119.
  9. Olstad, E. W., Ringers, C., Hansen, J. N., Wens, A., Brandt, C., Wachten, D., Yaksi, E. and Jurisch-Yaksi, N. (2019). Ciliary beating compartmentalizes cerebrospinal fluid flow in the brain and regulates ventricular development. Curr Biol 29(2): 229-241 e226.
  10. Satir, P. and Sleigh, M. A. (1990). The physiology of cilia and mucociliary interactions. Annu Rev Physiol 52: 137-155.
  11. Yuan, S., Liu, Y., Peng, H., Tang, C., Hennig, G. W., Wang, Z., Wang, L., Yu, T., Klukovich, R., Zhang, Y., Zheng, H., Xu, C., Wu, J., Hess, R. A. and Yan, W. (2019). Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence. Proc Natl Acad Sci U S A 116(9): 3584-3593.

简介

[摘要 ] 体外生物物理测量为了解生理和致病过程提供了宝贵的见识。由这些生物物理特性调节的一种关键的生理机制是纤毛产生的血流,它介导了粘膜纤毛清除,已知它可以抵抗上呼吸道中的异物和病原体。为了测量睫毛清除率,已实施了多种技术,包括使用放射性标记的颗粒和使用单光子发射计算机断层扫描(SPECT)方法进行成像。这些测试虽然是非侵入性的,但需要使用专用设备,从而限制了其广泛使用。这里我们描述一种离体方法 对纤毛产生的血流进行成像,并采用以前报道的方法进行改编,以使研究人员更易于访问并获得更高的通量。我们在安乐死后迅速从小鼠身上切除气管,将其纵向切割并将其放置在内部制作的幻灯片中。我们应用荧光颗粒在荧光显微镜下测量颗粒的运动,然后使用ImageJ进行分析,从而可以计算不同条件下纤毛产生的流体流量。该方法能够以最少的投资或专用设备进行组织中的离体测量,从而有机会研究和发现与气管的睫状运动在生理和疾病方面相关的重要生物物理特性。

[背景 ] 多样的生物物理特性的组合物,每个辅助指定功能,弥补人体。一个重要的例子是在许多不同器官系统中产生血流,包括中枢神经系统(Olstad 等人,2019),生殖道(Afzelius 等人,1978; Yuan 等人,2019)和肺系统(Satir和Sleigh,1990),由专门的纤毛细胞制成。了解环境或外部刺激如何影响这些过程可以使我们深入了解疾病。在我们最近的研究中(Kudo 等人,2019),我们探索了湿度如何影响小鼠模型中的流感感染。通过使用这种离体测量小鼠纤毛产生的血流的方法,我们能够直观地测量环境湿度如何改变病毒清除率(图1和2)。先前的研究已经使用放射性标记的颗粒通过无创方法测量粘膜纤毛清除率(Hua 等人,2010; Bustamante-Marin和Ostrowski,2017)。但是,这需要使用放射性物质和SPECT或其他机器来确定可能不容易获得的放射性。这样的测量可以使用更多的人使用前VIV Ó 的成像在组织颗粒(南切。等人,2012;弗朗西斯和Lo,2013; Mastorakos 等人,2015和2016) 。在这里,我们描述了一种测量纤毛在上呼吸道组织中产生的流量的详细方法,改编自Francis和Lo (2013)。我们的方法易于执行,并允许以相对较低的成本测量大量的小鼠。该协议也可以在短时间内执行,从而可以保留大量动物的生物学差异并进行测量。最后,它可以用于其他器官系统或组织,测量各种生物物理特性,如Nance 等人所述。(2012年),在确定哪些标准必须满足载药颗粒穿越脑实质。总之,该协议将为研究人员提供一种易于遵循的分步方法,以离体测量生物组织的特性(图3)。

关键字:纤毛, 生物物理, 黏膜纤毛清除, 肺, 离体成像

材料和试剂


 


Superfrost Plus金片(Thermo Fisher Scientific,目录号:FT4981GLPLUS)。品牌并不重要
方盖滑片(Thermo F isher Scientific,目录号:18X18-1)。品牌并不重要
电工胶带(3 M Safety,Scotch,在亚马逊购买)。品牌并不重要
FluoSpheres 羧酸乙酯,0.2 微米绯红,645分之625(Life Technologies公司,目录号:F8806)替代尺寸/可用于化学取决于实验设计,请参考上述背景
6 - 孔板(Fisher Scientific公司,Corning)中。品牌不是关键,不必是组织培养质量
老鼠
PBS(Millipore Sigma,目录号:D8537)。品牌并不重要
超级胶水(乐泰,在亚马逊购买)。品牌不重要
PBS中的FluoSpheres 羧酸盐的体积比为1:1,000 (请参阅食谱)
 


设备


 


解剖剪刀(Fine Science Tools,目录号:14061-10)品牌并不重要
电动剃须刀(从亚马逊购买)的品牌并不重要
脱毛产品Nair(从亚马逊购买)品牌并不重要
镊子(精细科学工具)品牌并不重要
显微解剖剪刀(精细科学工具)品牌并不重要
手术刀(从亚马逊购买)可选,品牌并不重要
解剖显微镜(Laxco ,LMS-Z200)可选,品牌并不重要
布鲁克Opterra 扫频显微镜(布鲁克)的品牌并不重要,显微镜必须配备高速荧光摄像头(Photometrics Evolve Delta EMCCD摄像头)




软件


 


1. 获取图像Prairie View 5.4(布鲁克)品牌并不重要,任何与显微镜相关的软件都可以使用       


2. 分析软件ImageJ(方向性,Mtrack2,手动跟踪,NIH,https: //imagej.nih.gov/ij/ )可以使用其他软件进行分析       


 


程序


 


准备幻灯片
制作一堆电工胶带,使其厚度约为1.5毫米(7根胶带)(图1A-1B)。
注意:此处的胶带厚度可能会根据您要处理的薄纸的厚度而有所不同,其厚度对应于薄纸接触玻片底部和盖玻片的底部而不会明显扭曲薄纸。但是,重要的是要使样本之间的厚度保持一致,因为厚度会在以后影响成像。


使用手术刀切一小段,以容纳气管(图1C)。
将胶带放在载玻片上,制成可用于大多数显微镜的容纳腔的载玻片(图1D)。
注意:实验前可以轻松准备大量幻灯片。这样做可以加快实验的通量。


 






图1. 准备用于测量粘膜纤毛间隙的小室玻片。AB 。电工胶带彼此堆叠在一起,形成相当于〜1.5 mm的堆叠(白色箭头)。C. 在带子的中央切开部分以形成用于组织的腔室。D.将胶带粘贴到载玻片上以进行成像。E. 气管组织的例子,其被纵向切割并以盖玻片放置在腔室中。


 


气管夹层(时间敏感)
遵循以下步骤,直到对每只小鼠不间断地进行图像采集为止(而不是为实验提取所有气管),并迅速获得最佳结果。


根据当地机构动物护理和使用委员会(IACUC)的政策和协议对小鼠实施安乐死。
使用解剖板或将鼠标拍打在坚硬的表面上来固定鼠标。立即使用您的选择(奈尔,电动剃须刀)的脱毛产品暴露颈部皮肤,使一〜3 从胸部到鼻子厘米的切口,并移动唾液腺一边,露出小鼠的气管。
清洁与气管相关的结缔组织,并在气管上端进行横切。用镊子握住气管以保持气管紧绷。
切开气管下端。
立即传送到6孔板约1 米升PBS。
使用显微解剖剪刀从气管纵向切开。
立即继续进行下一步。
 


安装气管
              准备PBS 中1:1,000的FluoSpheres 羧酸盐溶液。
注意:请准备大量用于整个实验。充分涡旋初始溶液,并在每次使用之间充分涡旋混合。


将步骤B的气管放入步骤A的玻片中,管腔一侧朝上。
注意:使胶带的高度与组织的厚度相匹配是至关重要的,因为这样可以使盖玻片防止在成像过程中气管重新折叠。


地方〜250 μ 的1升:1,000 FluoSphere 在腔室的溶液,放置超级胶水几滴磁带和盖玻片(图的边缘1E)。
注意:请注意不要在腔室中放入过多的液体,也不要在胶带上放置过多的胶水。目的是要有足够的液体将样品浸没或完全覆盖中心而不会溢出。将浸没在样品中的液体与超级胶水混合会导致结果不可靠。使用练习幻灯片进行优化,以获取每种设置所需的确切音量。


立即继续进行下一步。
 


影像学
              显微镜应打开并设置在正确的位置。使用含有颗粒溶液但无器官的玻片作为阳性对照(请参阅视频1)。
注意:成像是在室温下进行的。ħ H但是,实验可以在不同温度下,如果您的显微镜装置进行允许不同的温度设置。


 






视频1. 粒子在自由溶液中以布朗运动运动的示例


 


将幻灯片放到位。
*重要*更改焦点,直到找到一个平面,其中的粒子似乎是静态的。这是组织的平面,其中颗粒附着/吞噬到表面上的粘液/细胞。从那里开始,将焦点移至每个图像的设定量。这一点很重要,因为纤毛产生的流量的测量值会随着距纤毛表面的距离而变化。因此,要生成可以在样本之间进行比较的数据,必须将该距离保持恒定(请参阅视频2)。在我们的实验中,我们使用的距离的〜1 μ 从纤毛的表面米的距离。
 






视频2.由于纤毛产生的流,粒子按方向运动的示例


 


以每秒100帧或以上的速度s 采集图像(我们以100 fps的速度采集了30 s)。用显微镜设置拍摄图像;图像尺寸:512 μ 米X 512 μ 米,FOV:204.2 μ 米X 204.2 μ m和像素尺寸0.399 μ 米×0.399 μ 米。
注意:我们实验中的两个成像激光器分别为488 nm和561 nm,其发射滤光片针对GFP和RFP进行了优化。两个照相机在显微镜AR Ë512 μ 米X 512 μ 中号光曲线演变德尔塔EMCCD相机。激光是通过狭缝或32个针孔阵列进行扫描的,旨在提高扫描速度,全帧时max速度为67 fps ,并具有较小的ROI,可实现更快的速度。


 


尽快拍摄这些图像至关重要。在我们的手中,所有图像均在解剖后5分钟内拍摄,并在10分钟后看到组织活力降低。
注意:此步骤的关键部分是使操作过程舒适并减少解剖和成像之间的时间。练习获得良好的流动,或成对工作以让一个人解剖,而另一个人则要设置显微镜以建立最佳成像条件。作为纤毛运动调节的积极对照,环境湿度较高(〜50 %)的小鼠将表现出较高的纤毛颗粒速度(Kudo等人,2019)。作为阴性对照,可以使用环境湿度较低的小鼠,也可以使用经过较长时间(> 10分钟)切除的气管。


 


数据分析


 


使用文件>导入>图像序列将数据加载到FIJI / ImageJ中。
使用插件>跟踪> MTrack2(https://imagej.net/MTrack2)分析粒子速度。
注意:使用imageJ 设置“图像”>“调整”>“亮度/对比度”以确保高信噪比。这将使MTrack2程序可以更好地识别粒子。


使用插件>跟踪>手动跟踪(https://imagej.nih.gov/ij/plugins/track/track.html)测量单个粒子的轨迹(图2)。
注意:此功能将允许记录单个粒子的坐标。然后可以使用绘图软件对其进行绘图,以显示给定时间内粒子的整个轨迹。


使用“分析”>“方向性”来计算流的方向性(图3 )。
使用文件>另存为> A VI 导出粒子流的视频(视频1和2)版本。
 






图2. 纤毛产生的流量和方向性分析V IDEOS 1和2 中A.定量纤毛生成的流的V IDEO 小号1和2中使用的数据一个协议的nalysis部分。公元前。V ideo s 1和2 的方向性频率的量化。


 






图3. 纤毛产生流量测量的主要步骤示意图


 


菜谱


 


1:1,000体积/体积的FluoSpheres 羧酸盐在PBS中
解决方案应存放在黑暗中


每天制作新鲜溶液,并在使用前平衡至室温


 


致谢


 


感谢Melissa Linehan的技术和后勤帮助。感谢Al Mennone 为这些实验设置显微镜,并感谢工藤惠子(Eriko Kudo)进行了导致产生原始研究论文的实验。图形插图由Biorender.com制作。这项工作得到了霍华德·休斯医学研究所(AI)的部分支持,这是康德尔集团(Condair Group)和美国国立卫生研究院(National Institutes of Health)赠款T32GM007205(对ES的医学科学家培训计划培训)提供的礼物。该协议的原始研究论文可以在Kudo 等人找到。(2019)。


 


利益争夺


 


作者对此书中描述的协议没有任何财务和非财务方面的竞争利益。






伦理


 


本研究未使用任何人类受试者。根据Yale IACUC协议2018-10365批准了所有动物工作。


 


参考文献


 


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引用:Song, E. and Iwasaki, A. (2020). Method for Measuring Mucociliary Clearance and Cilia-generated Flow in Mice by ex vivo Imaging. Bio-protocol 10(6): e3554. DOI: 10.21769/BioProtoc.3554.
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