Summary

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

Published: May 14, 2016
doi:

Summary

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Abstract

单细胞对抗癌药物的响应中确定人口响应显著贡献,并因此是在整体结果的主要因素。免疫印迹,流式细胞术和固定细胞实验经常用于研究细胞的抗癌药物的反应。这些方法是重要的,但是它们具有若干缺点。变异药物反应癌细胞与正常细胞之间的,和瞬态响应罕见的癌症不同来源的细胞,之间是很难理解用人口平均测定,不能够直接跟踪和纵向分析它们。显微镜特别适合于图像的活细胞。技术的进步使我们能够在一个分辨率不仅能细胞跟踪,而且多种细胞反应观察常规图像的细胞。我们详细描述了一种方法,允许的连续时间推移成像为基本上只要需要,典型地高达96小时的药物的反应过程中的细胞。使用该方法的变型中,细胞可以为周进行监测。随着就业的基因编码荧光生物传感器大量的进程,途径和应答可以遵循。我们表明的例子,其中包括跟踪和细胞生长和细胞周期进程,染色体动力学,DNA损伤的定量,和细胞死亡。我们还讨论技术和灵活性的变化,并突出显示一些常见的陷阱。

Introduction

活细胞显微镜和单细胞的纵向跟踪不是一个新的技术。从最早的显微镜,爱好者和科学家们观察和研究单个细胞和生物体,他们的行为和发展1-3。从已故的大卫·罗杰斯在20世纪50年代范德比尔特大学一个著名的例子显示了血涂片追金黄色葡萄球菌和吞噬4的最终过程中的人中性粒细胞。此活细胞电影是如何多个进程可以观察和相关的单次实验优异的例证:化学梯度,力学和细胞运动的速度的感测,细胞形状动力学,粘附和病原体的吞噬作用。

全自动显微镜和高度敏感的数码相机的出现,导致了使用显微镜调查的越来越多问细胞生物学范围˚F基本问题ROM细胞如何移动和5,67,8细胞器的动态和膜贩运9-11。无荧光,明场显微镜,包括相位对比(PC),它囊括了诺贝尔奖为弗里茨·泽尔尼克于1953年,和微分干涉对比(DIC)允许细胞和细胞核,而且亚细胞结构,包括微管束的观察,染色体,核仁,细胞器动力学和粗纤维肌动蛋白12。基因编码荧光蛋白和细胞器对荧光染料的发展已经大大影响时间推移显微镜13-15。尽管本文的不关注的焦点,在细胞球体和使用共聚焦和多光子显微镜原位 (活体显微镜)成像代表了该方法的另一个扩张,并有使用和讨论这些方法16-19优秀物品。

细胞的抗CANC响应呃药物或天然产物的分子和细胞比例来确定。理解细胞应答和命运以下治疗常常涉及人口平均测定( 例如 ,免疫印迹,全井措施),或与免疫荧光检测固定时间点和流式细胞仪,用来测量单细胞。异质性群体内单个细胞应答的药物,特别是在肿瘤中,可以解释一些变异的响应跨越了与在饱和相同药物处理的细胞系和肿瘤中看到。长期纵向方法遵循给定的单细胞或细胞群是不常见的,但非常强大的方法,其允许分子响应途 ​​径的直接研究,不同表型( 例如 ,细胞死亡或细胞分裂),观察细胞至细胞变异的群体内,以及如何将这些因素有助于人口响应动力学20-22。乐观,能够观察和量化的单细胞反应将有助于改善我们的药是如何工作的,为什么他们有时会失败,以及如何最好地使用他们的理解。

长期时间推移显微镜,纵向跟踪,以及药物反应分析的技术可用于许多研究者,并且可以是简单的,只用透射光观察的表型反应20,21。该方法的主要组成部分包括:感兴趣的细胞的适当的制剂,用环境室的自动显微镜,照相机与计算机集成的获取和存储图像,以及软件审查时间推移和测量和分析细胞和任何荧光生物传感器。我们提供了一个详细的协议,它为使用明和/或广角镜啶进行培养细胞的时间推移显微镜,只要几天许多技巧。该协议可以用于任何细胞系,可以在培养物中生长研究其抗癌治疗的反应。我们提供获得的数据的实施例和使用多个不同的基因编码的荧光生物传感器和相衬显微镜的一例,简要地讨论的不同类型的探针,优点和长期的时间推移和纵向跟踪的缺点进行了分析,什么都可以了解到这种方法,很难从非直接方法,以及一些变化,我们希望将有兴趣和价值经验的研究人员使用的方法谁没有考虑到理解,以及经验丰富的研究。

Protocol

以下方案使用由实验在图4 和6关于采集设置和实验条件定义的参数。许多这些参数可以被修改以适应其它实验( 即,曝光时间,分级,荧光通道等 )。所有的程序必须坚持体制准则和条例,并通过生物安全委员会的批准。显微镜制造商的网站含有活细胞成像优秀的信息。 1.显微镜和图像处理软件在各种各样的倒置显微镜进行活?…

Representative Results

长期时间推移显微镜和直接纵向跟踪允许许多抗癌作用药物的反应在研究。以下图1中的大致轮廓,示出细胞的多个例子表达验证荧光记者,具有抗癌药物,追踪处理,并使用不同的方法进行分析。 单独相衬显微镜是非常翔实和有力的间期与有丝分裂,有丝分裂期和停滞,不正常的细胞分裂和细胞死亡21,23,24</su…

Discussion

时间推移显微镜和纵向跟踪的优势

该显微镜是用于药物反应的纵向研究的理想工具,因为它允许调查者跟踪单个细胞和他们的命运以及整个人口。变异的细胞群内的药物反应是用于抗癌治疗设计的一个主要问题。单个细胞的纵向跟踪调查可以观察到这种变化,并开始了解的底层机制和后果,因为它涉及到细胞群。利用各种荧光探针提供的方法来观察和了解双方?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and β-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Academy of Sciences, Austria).

Materials

Taxol (paclitaxel) Sigma T7191 microtubule stabilizing drug
etoposide Selleckchem S1225 topoisomerase II inhibitor
selinexor Karyopharm Therapeutics na XPO1/CRM1 inhibitor, gift
Kinesin-5 inhibitor Merck Serono na gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2
cell growth medium HyClone (Fisher) or Mediatech many companies available
5% CO2/balance air, certified Airgas Z03NI7222004379
35mm dish, 20mm glass bottom Cellvis D35-20-1.5-N many companies available
35mm 4 well dish, 20mm glass bottom Cellvis D35C4-20-1.5-N many companies available
35mm dish, gridded glass bottom MatTek P35G-2-14-CGRD many companies available
multi-well, glass bottom Cellvis P12-1.5H-N many companies available
Olympus IX81 inverted epifluorescence microscope Olympus
Olympus IX2-UCB controller Olympus
PRIOR LumenPro200 Prior Scientific Lumen200PRO
PRIOR Proscan III motorized stage Prio Scientific H117
STEV chamber InVivo Scientific STEV.ECU.HC5 STAGE TOP
Environmental Controller Unit InVivo Scientific STEV.ECU.HC5 STAGE TOP
Hamamatsu ORCA R2 CCD with controller Hamamatsu C10600
Nikon Eclipse Ti Nikon
Nikon laser launch Nikon
SOLA light engine lumencor
iXon Ultra 897 EM-CCD ANDOR 
TOKAI HIT inclubation chamber TOKAI HIT TIZSH

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Cite This Article
Burke, R. T., Orth, J. D. Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics. J. Vis. Exp. (111), e53994, doi:10.3791/53994 (2016).

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