Szatmary Alex C, Stuelten Christina H, Nossal Ralph
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health, and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
RSC Adv. 2014 Nov 5;4(100):57343-57349. doi: 10.1039/C4RA08572H.
Migration of cells along gradients of effector molecules, i.e., chemotaxis, is necessary in immune response and is involved in development and cancer metastasis. The experimental assessment of chemotaxis thus is of high interest. The agarose spot assay is a simple tissue culture system used to analyze chemotaxis. Although direction sensing requires gradients to be sufficiently steep, how the chemical gradients developed in this assay change over time, and thus, under what conditions chemotaxis is plausible, has not yet been determined. Here, we use numerical solution of the diffusion equation to determine the chemoattractant gradient produced in the assay. Our analysis shows that, for the usual spot size, the lifetime of the assay is optimized if the chemoattractant concentration in the spot is initially 30 times the dissociation constant of the chemoattractant-receptor bond. This result holds regardless of the properties of the chemoattractant. With this initial concentration, the chemoattractant gradient falls to the minimum threshold for directional sensing at the same time that the concentration drops to the optimal level for detecting gradient direction. If a higher initial chemoattractant concentration is used, the useful lifetime of the assay is likely to be shortened because receptor saturation may decrease the cells' sensitivity to the gradient; lower initial concentrations would result in too little chemoattractant for the cells to detect. Moreover, chemoattractants with higher diffusion coefficients would sustain gradients for less time. Based on previous measurements of the diffusion coefficients of the chemoattractants EGF and CXCL12, we estimate that the assay will produce gradients that cells can sense for a duration of 10 h for EGF and 5 h for CXCL12. These gradient durations are comparable to what can be achieved with the Boyden chamber assay. The analysis presented in this work facilitates determination of suitable parameters for the assay, and can be used to assess whether observed cell motility is likely due to chemotaxis or chemokinesis.
细胞沿效应分子梯度迁移，即趋化作用，在免疫反应中是必需的，并且参与发育和癌症转移。因此，趋化作用的实验评估备受关注。琼脂糖斑点试验是一种用于分析趋化作用的简单组织培养系统。尽管方向感知需要梯度足够陡峭，但该试验中产生的化学梯度如何随时间变化，以及因此在何种条件下趋化作用是合理的，尚未确定。在这里，我们使用扩散方程的数值解来确定试验中产生的趋化因子梯度。我们的分析表明，对于通常的斑点大小，如果斑点中趋化因子的初始浓度是趋化因子 - 受体键解离常数的30倍，则试验的持续时间将得到优化。无论趋化因子的性质如何，该结果均成立。在这个初始浓度下，趋化因子梯度降至方向感知的最小阈值的同时，浓度也降至检测梯度方向的最佳水平。如果使用更高的初始趋化因子浓度，试验的有效持续时间可能会缩短，因为受体饱和可能会降低细胞对梯度的敏感性；较低的初始浓度会导致细胞可检测的趋化因子过少。此外，具有较高扩散系数的趋化因子维持梯度的时间会更短。基于先前对趋化因子表皮生长因子（EGF）和CXC趋化因子配体12（CXCL12）扩散系数的测量，我们估计该试验将产生细胞能够感知10小时的EGF梯度和5小时的CXCL12梯度。这些梯度持续时间与Boyden小室试验所能达到的相当。这项工作中提出的分析有助于确定试验的合适参数，并可用于评估观察到的细胞运动是否可能是由于趋化作用或化学增活作用。
