成像效应在耳朵上的记忆体T细胞诱导后收养的DTH

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Summary

在这里,我们证明了诱导和记录一个迟发型超敏反应(DTH)反应在鼠耳进展的方法。这是由鼠耳组织编制的示范效应/记忆性T细胞反应的双光子成像。

Cite this Article

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Matheu, M. P., Beeton, C., Parker, I., Chandy, K. G., D. Cahalan, M. Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH. J. Vis. Exp. (18), e907, doi:10.3791/907 (2008).

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Abstract

迟发型超敏反应(DTH)是主体CCR7的-效应/记忆性T淋巴细胞免疫反应。在这里,我们展示了一种诱导和记录在大鼠耳的DTH反应的进展的方法。这是由双光子成像的CCR7的示范-效应/记忆T细胞应答鼠耳组织编制。

收养的DTH诱导GFP标记的OVA特定CCR7的腹腔注射 -效应/记忆T细胞系(Beeton,C研究的可视化实验,第8期) 。然后让细胞在大鼠平衡,1:1混合的卵子和卵子的共轭得克萨斯红(OVA - TR)的一只耳朵注射用生理盐水(对照耳)和其他允许可视化挑战48小时前居民的抗原提呈细胞。

我们描述了一个有用成像结合胶原纤维的二次谐波产生的可视化过程中的免疫反应,深层的真皮层内的细胞活力的组织准备的方法。耳组织切成5 × 5毫米平方(稍大更好),装到塑料盖使用Vetbond™单,然后安全的使用在成像室硅脂和37氧冒泡的组织培养液灌流° C 。

Protocol

收养的DTH诱导

请看到的Jove文章收养大鼠迟发型超敏反应的感应和监测
恭Beeton,乔治K。Chandy
生理学和生物物理学系,美国加州大学欧文分校
可视化实验研究,第8期

在这里,我们使用1:1的比例与未​​标记的抗原的抗原(卵清蛋白)结合德州红,让吞噬抗原提呈细胞的可视化修改这一协议。

耳组织收获

  1. 安乐死在批准的动物协议中列出的程序所需的时间点,大鼠。在这里,我们使用过量的麻醉,异氟醚。这是在位于通风良好的引擎盖内的密闭容器中。确保动物是要么打开胸腔,切心斩首的死。

  2. 使用大剪刀移除一个单一的切割,尽可能接近动物体内(图1)耳。

    Figure_1.jpg
    图1。去除鼠耳

  3. 放置在一个含有冰冷的1X PBS或RPMI - 1640〜10毫升15毫升锥形管耳。置于冰上,直到您准备,以备成像组织的组织样本。

  4. 图片组织尽快,我们发现,虽然1至2小时在冰上,在组织没有可辨别的影响细胞的运动相比,收获和成像立即组织。

成像的组织准备

一,去除表皮和真皮层

*注 :深真皮层,必须保持完整。如果做得正确,大血管,将保持不变,耳朵软骨不会被暴露。这是一个很难的技术,特别是当有没有炎症控制条件和使用解剖显微镜,它是有帮助的。

  1. 将组织从10 cm组织培养皿中的锥形管和媒体。

  2. 带着手套的双手抱大鼠耳边轻轻耳朝上(图2)耳背侧(远端)的一角。

    Figure_2.jpg
    图2。修剪皮毛断耳

  3. 用你的拇指之上的耳朵,稳定的组织,而使用封闭杜蒙提示#5剪刀分开真皮深层真皮。另外,如果耳朵边缘的皮肤/真皮深层悬,这可以掌握用镊子轻轻拉分开,从下面的组织(图3)。

    Figure_3.jpg
    图3。去除真皮上部

  4. 一旦小面积的皮肤已经从底层组织分开,重复的分离步骤,或者使用之间的真皮层和镊子剪刀削减轻轻地清除和剥离脱落的皮肤。

  5. 揭露一个5 x 5毫米或更大的耳组织,从最初的抗原注射网站(图4)至少有3毫米的面积。

    Figure_4.jpg
    图4。组织成像暴露

二。保护组织的成像阶段

  1. 剪下一个稍大比准备组织大小的塑料盖滑。

  2. 传播3M Vetbond™安全组织跨幻灯片胶薄薄的一层。

  3. 抓住准备组织(你是不是有可能形象)的角落,从媒体中删除,并组织民建联的镜头纸或许姆底面擦拭,除去多余的介质。

  4. 触摸胶水覆盖的幻灯片(湿纸巾立即Vetbond治愈)组织的边缘。轻轻地舒展组织扁平,最后触摸幻灯片(图5)与异性结束

    Figure_5.jpg
    图5。摩组织盖玻片

  5. 固化Vetbond™反相组织样本/幻灯片和水库的媒体,如组织和幻灯片在45度角朝下,大约浸泡。倒挂控股的角度组织,有利于暴露组织表面固化,以防止多余的胶水。组织表面上胶块激光(图6)。

    Figure_6.jpg
    图6。固化vetbond组织GLUE的媒体浸渍

    注意 :步骤2-5可能需要稍加练习掌握。先试后首次试图组织块丢弃或额外练习。

  6. 民建联的少量硅脂到幻灯片的底部。

  7. 放置在灌注室的幻灯片。确保灌注媒体是流动的,37℃,并加入组织前含氧。轻轻向下按幻灯片灌注腔的底部和塑料盖滑之间的硅脂密封边缘。

三。双光子成像

  1. 使用明场透射光,着眼于组织的顶部。关闭所有的灯。

  2. 打开激光,光电倍增管所要求的多光子显微镜系统。

  3. 高度有序的纤维蛋白二次谐波(SHG)(胶原蛋白和肌球蛋白)产生的信号在激发光的波长的一半。我们使用900纳米的激发,导致在450 nm处,它可以可视化使用“蓝色”通道显微镜倍频。倍频一代,是一种非线性的(双光子)的过程中,从而提供了光学切片“双光子激发荧光效果相似。倍频峰效率时,会发生纤维垂直于激光并行运行的纤维,而不会被显示。

  4. 用大量的细胞中找到一个很好的成像面积,购置面积等,多数细胞在中心,并开始成像设置。注:在DTH反应,T细胞及其他细胞浸润经常发现被集中到了一起,而其他地区都没有浸润细胞 2 。因此,它可能需要一些时间,寻找组织,找到最好的地区形象。

  5. 检查评估期间的成像速度和极性细胞细胞活力。使用最低的激光功率,提供可接受的图像质量,以确保最小的光损伤。

  6. 交换需要一个新的组织准备。

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References

  1. Koo, G. C., et al. Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo. J Immunol. 158, 5120-5128 (1997).
  2. Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity. J Immunol. 147, 816-822 (1991).
  3. Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy. J Neuroimmunol. 191, 86-97 (2007).
  4. Kawakami, N., et al. Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med. 201, 1805-1814 (2005).
  5. Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science. 296, 1869-1873 (2002).
  6. Matheu, M. P. B., A, C. G. arcia, Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block. Immunity. In Press (2008).

Comments

6 Comments

  1. Hello. That was a great explanation for adoptive transfer and the techniques associated with that. In our laboratory, we are trying to employ DTH mouse model but we are using footpad, instead of an ear as it was shown that ear is less sensitive to linked suppression that footpad (W. J. Burlingham et. al.) We just acquired the same micrometer (thickness gauge) but our reading are very inconsistent. I was wondering if you ever tried footpad and could share some methods/hints to get stable/reproducible measurements.  Thanks, Vyacheslav Palchevskiy, Ph.D.

    Reply
    Posted by: Anonymous
    November 3, 2008 - 10:27 PM
  2. Dear Vyacheslav, we have done a few trials in the footpad and have also had difficulties getting consistent results. I think it is because this area contains more soft tissue than the ear. One thing that may help: make sure your injections are very consistent in terms of amount and location (including depth). We have found that the depth of injection can considerably change the amount of inflammation and the number of T cells in the tissue. This is not a problem in the ear as you can’t really go too deep there. We always take at least 6 serial measurements for each ear or paw and then calculate a mean to try and get rid of the variation. The more variation we have, the more measurements we take of each ear or paw. Hope this help! Christine

    Reply
    Posted by: Christine B.
    November 11, 2008 - 4:52 PM
  3. We are in agreement  that the mouse footpad presents technical problems, even for routine histology. We use footpad for adoptive transfer of human PBMC because this site seems more sensitive for detecting regulation; however if Vyacheslav only wants to study effector response the mouse ear works fine. Your imaging system looks very appealing . We will try in ear first, then later in footpad. Thanks for this very nice 'webinar'. Cheers--Will Burlingham, UW-Madison, WI

    Reply
    Posted by: Anonymous
    December 5, 2008 - 3:41 PM
  4. Christine, I see you are using an Olympus ²0x dipping lens--is your ²photon system breadboard or a commercial Olympus? Right after you focus the objective into the ear tissue, the video switchs to the fluorescence time lapse.  Do you cover your sample/microscope or is your room very very dark.  Are you using non-descanned detectors?  Reason I am asking is on my ²004 BioRad MP system I have to cover the sample, monitors etc in order to use the nondesanned detectors.  It is hard for me to try these studies as I am also on an inverted system.  What wavelength are you using? Lance Rodenkirch UW

    Reply
    Posted by: Anonymous
    December 10, 2008 - 5:13 PM
  5. Hi Lance, This is Melanie. We use a custom breadboard for our two photon scan head system, the detailed plans for our custom system are on the Parker Laboratory (UC Irvine) website. We both keep the room dark and cover our microscope and PMT detectors with black-out curtain to reduce noise from stray light. In order to manipulate the sample after this we work "blind" with our hands under the black-out curtain. Our PMT detectors are also mounted inside sealed aluminum box. To reduce stray light from the monitors from affecting the sample we have them at an angle facing slightly away from where the microscope and PMTs are, but we do not cover them. To visualize collagen and GFP at the same time we use between 890 and 9²0nM. 9²0nM is typically the wavelength we used. I hope this helps. Do check the Parker Lab website for more details on the system. Best of luck with your experiments! Melanie  

    Reply
    Posted by: Melanie M.
    January 20, 2009 - 11:29 AM
  6. Great demo. We can employ this to our Imm. Lab. for the medical students. Can we acquire reagents from you girls and try it out here in Mackay Medical College, Taiwan.

    Reply
    Posted by: Nan Chi C.
    April 20, 2011 - 5:46 PM

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