High-throughput assays show the timescale for phagocytic success depends on the target toughness
High-throughput assays show the timescale for phagocytic success depends on the target toughness
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DOI：
10.1063/5.0057071
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发表时间：
2021-09-01
期刊：
影响因子：
--
通讯作者：
Gordon, Vernita D.
中科院分区：
文献类型：
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作者：
Bakhtiari, Layla A.;Wells, Marilyn J.;Gordon, Vernita D.
吞噬免疫细胞可以通过吞噬病原体来清除体内的病原体。细菌生物膜是在基质中结合在一起的细菌群落，所述基质赋予生物膜粘弹性机械性质，所述粘弹性机械性质对于自由游动的细菌不存在。由于中性粒细胞太小，无法吞噬整个生物膜，因此如果要利用吞噬作用清除感染，它必须能够一次分离并吞噬一些细菌。我们最近发现，目标弹性和吞噬成功之间的负相关性。早期的工作使用耗时的人工分析中性粒细胞和荧光珠的显微照片。在这里，我们介绍并验证流式细胞术作为一种快速，高通量的技术，增加了两个数量级的实验分析的中性粒细胞的数量，同时也减少了所需的时间，这样做从几小时到几分钟。我们还介绍了使用聚丙烯酰胺凝胶在我们的测定吞噬成功。聚丙烯酰胺凝胶的可调性扩展了我们可以研究的机械参数空间，并且我们发现，即使具有低弹性，高韧性和屈服应变也会影响吞噬成功及其时间尺度。对于刚性凝胶与低产量的应变，以及随之而来的低韧性，吞噬的成功是近四倍，当中性粒细胞与凝胶孵育6小时后，只有1小时的孵育。相比之下，对于具有高屈服应变和随之而来的高韧性的软凝胶，成功的吞噬对时间的敏感性要小得多，从1小时到6小时的孵育增加了不到两倍。
Phagocytic immune cells can clear pathogens from the body by engulfing them. Bacterial biofilms are communities of bacteria that are bound together in a matrix that gives biofilms viscoelastic mechanical properties that do not exist for free-swimming bacteria. Since a neutrophil is too small to engulf an entire biofilm, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. We recently found a negative correlation between the target elasticity and phagocytic success. That earlier work used time-consuming, manual analysis of micrographs of neutrophils and fluorescent beads. Here, we introduce and validate flow cytometry as a fast and high-throughput technique that increases the number of neutrophils analyzed per experiment by two orders of magnitude, while also reducing the time required to do so from hours to minutes. We also introduce the use of polyacrylamide gels in our assay for engulfment success. The tunability of polyacrylamide gels expands the mechanical parameter space we can study, and we find that high toughness and yield strain, even with low elasticity, also impact the phagocytic success as well as the timescale thereof. For stiff gels with low-yield strain, and consequent low toughness, phagocytic success is nearly four times greater when neutrophils are incubated with gels for 6 h than after only 1 h of incubation. In contrast, for soft gels with high-yield strain and consequent high toughness, successful engulfment is much less time-sensitive, increasing by less than a factor of two from 1 to 6 h incubation.