Droplet encapsulation improves accuracy of immune cell cytokine capture assays
Droplet encapsulation improves accuracy of immune cell cytokine capture assays
复制标题
液滴封装提高了免疫细胞细胞因子捕获测定的准确性
DOI：
10.1039/c9lc01261c
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发表时间：
2020
期刊：
影响因子：
6.1
通讯作者：
Heyman, John A.
中科院分区：
文献类型：
--
作者：
Yuan, Yuan;Brouchon, Julie;Calvo-Calle, J. Mauricio;Xia, Jing;Sun, Li;Zhang, Xu;Clayton, Kiera L.;Ye, Fangfu;Weitz, David A.;Heyman, John A.
细胞分泌分子的定量，例如，细胞因子是表征免疫应答的基础。使用工程抗体将分泌的分子锚至分泌细胞的细胞因子捕获测定被广泛用于表征免疫应答，因为它们允许敏感地鉴定和回收活的应答细胞。然而，如果细胞因子扩散远离分泌细胞，则非分泌细胞也将被鉴定为应答细胞。在这里，我们将免疫细胞封装在微流体液滴中，并进行液滴内细胞因子捕获测定以限制分泌的细胞因子的扩散。我们使用微流控装置将单个自然杀伤NK-92 MI细胞及其靶细胞K562细胞快速封装到微流控液滴中。我们进行液滴内IFN-γ捕获测定，并证明NK-92 MI细胞识别液滴内的靶细胞并被激活以分泌IFN-γ。液滴包封防止分泌产物扩散到邻近细胞，并且相对于在没有液滴的情况下进行的测定，显著减少假阳性和假阴性。在含有1%真阳性的样品中，包封将显示为阴性的真阳性细胞的数量从94%减少到2%;在含有50%真阳性的样品中，显示为阳性的未刺激细胞的数量从98%减少到1%。在细胞从液滴释放后，分泌的细胞因子保持捕获在分泌免疫细胞上，使得能够对高度富集活化的效应免疫细胞的群体进行FACS分离。液滴包封可用于降低背景并改善任何单细胞分泌测定的检测。
Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay.