A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy
A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy
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DOI：
10.1371/journal.pone.0052251
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发表时间：
2012-12-19
期刊：
影响因子：
3.7
通讯作者：
Guido, Stefano
中科院分区：
文献类型：
--
作者：
Vasaturo, Angela;Caserta, Sergio;Guido, Stefano
细胞对化学梯度的定向反应，称为趋化性，在包括发育、免疫应答和肿瘤细胞侵袭的生理和病理过程中起重要作用。尽管有这样的影响，趋化性仍然是一个具有挑战性的过程，在体外生理相关的条件下进行研究，主要是由于在模拟体内环境的基质中产生良好表征和持续的梯度，同时允许动态细胞成像的困难。在这里，我们描述了一种新的趋化性测定在3D胶原蛋白凝胶，基于可重复使用的直视室，其中的趋化梯度通过扩散通过多孔膜产生。通过落射荧光显微镜监测FITC标记的右旋糖酐的浓度，并将实验数据与基于菲克定律的理论和数值预测进行比较，分析了扩散过程。细胞迁移向趋化梯度已其次是延时显微镜和定量的细胞跟踪图像分析技术的基础上。结果以趋化指数（I）和平均细胞速度表示。通过比较在各向同性条件下和存在白细胞介素-8（IL-8）梯度的情况下人中性粒细胞的迁移，对该测定法进行了检测。在不存在IL-8刺激的情况下，80%的细胞显示出0至1 mm/min的速度。然而，在存在IL-8梯度的情况下，60%的细胞显示出速度增加，达到2至7 mm/min之间的值。此外，加入IL-8后，I分别从0增加至0.25和0.25增加至0.5，两个捐献者的情况这些数据表明中性粒细胞朝向3D胶原基质中的IL-8梯度的明显定向迁移。本文所述的趋化性测定可以适用于其他细胞类型，并且可以作为生理学相关的方法来研究细胞在3D环境中响应于不同化学引诱物的定向运动。
The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 mm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 mm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.