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.

中文翻译：

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液滴封装提高了免疫细胞细胞因子捕获测定的准确性。

细胞分泌分子例如细胞因子的定量是免疫反应表征的基础。使用工程化抗体将分泌分子锚定到分泌细胞的细胞因子捕获测定被广泛用于表征免疫反应，因为它们可以灵敏地识别和恢复存活的反应细胞。然而，如果细胞因子从分泌细胞扩散开，非分泌细胞也将被识别为响应细胞。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。我们使用微流体装置将单个自然杀伤 NK-92 MI 细胞及其目标 K562 细胞快速封装到微流体液滴中。我们进行了液滴内 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。相对于没有液滴进行的测定，液滴封装可防止分泌产物扩散到邻近细胞，并显着减少假阳性和假阴性。在含有 1% 真阳性的样品中，封装将显示为阴性的真阳性细胞数量从 94% 减少到 2%；在含有 50% 真阳性的样本中，显示为阳性的未刺激细胞数量从 98% 减少到 1%。细胞从液滴中释放后，分泌的细胞因子仍被捕获到分泌的免疫细胞上，从而能够对高度富集激活的效应免疫细胞的群体进行 FACS 分离。液滴封装可用于减少背景并改善任何单细胞分泌测定的检测。