Abstract：
Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.
摘要：
细胞在很大程度上依赖于外源营养的摄取来生存，增长,和差异化。然而,在单细胞水平上量化小分子营养素的摄取是困难的。在这里，我们提出了一种使用反向电子需求狄尔斯阿尔德（IEDDA）化学研究活单细胞中营养吸收的新方法。我们已经用四嗪修饰了羧基荧光素-二乙酸酯-琥珀酰亚胺酯（CFSE）-一种猝灭的荧光团，可以与蛋白质共价反应，并且仅在酯酶活性后在细胞的胞质溶胶中打开。该四嗪用作侧基荧光团的第二猝灭剂。在IEDDA反应中与富含电子或应变亲二烯体修饰的营养素反应后，这个淬火群被摧毁了，从而使探针发出荧光。这使得我们能够使用流式细胞术和活细胞显微镜监测活的初级免疫细胞群体中多种含亲双烯体营养素的摄取。
