Reif Randall D, Martinez Michelle M, Wang Kelong, Pappas Dimitri
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA.
Anal Bioanal Chem. 2009 Oct;395(3):787-95. doi: 10.1007/s00216-009-3024-1. Epub 2009 Aug 16.
A microfluidic device is designed and demonstrated for the simultaneous capture and induction of apoptosis in Jurkat cells. In this unique case, the cell capture event initiates a biological process. The device features a single channel made from poly(dimethylsiloxane) sealed to a glass substrate. The channel is coated with a series of reagents used in affinity chromatography separations of cells. In this case, the antibody used to capture the cells is functional anti-CD95 which captures the cells by binding to the Fas receptor on the cell membrane and, at the same time, inducing apoptosis via the caspase 8 pathway. Cells retained on the surface of the channel are known to be induced to undergo apoptosis. Medium is flowed slowly through the channel to maintain cell viability while the cells undergo apoptosis. After 3 h, staining with Annexin V-PE and 7-AAD revealed that 43.5% of the cells bound to the anti-CD95 coated channel are apoptotic, whereas 7.9% of cultured Jurkat cells induced with anti-CD95 for 3 h and stained in the same way were determined to be apoptotic by flow cytometry. The device provides a method of determining when apoptosis is induced, maintaining cell viability for long-term analysis and observing cells in real time as they are exposed to reagents that affect apoptosis. In the future, the device will be an invaluable tool for the study of the temporal dynamics of apoptosis.
设计并展示了一种用于同时捕获和诱导Jurkat细胞凋亡的微流控装置。在这种独特的情况下,细胞捕获事件启动了一个生物学过程。该装置的特点是有一个由聚二甲基硅氧烷制成的单通道,密封在玻璃基板上。通道涂有一系列用于细胞亲和色谱分离的试剂。在这种情况下,用于捕获细胞的抗体是功能性抗CD95,它通过与细胞膜上的Fas受体结合来捕获细胞,同时通过半胱天冬酶8途径诱导细胞凋亡。已知保留在通道表面的细胞会被诱导发生凋亡。培养基缓慢流过通道以维持细胞活力,同时细胞发生凋亡。3小时后,用膜联蛋白V-PE和7-AAD染色显示,与抗CD95包被通道结合的细胞中有43.5%发生凋亡,而用抗CD95诱导3小时并以相同方式染色的培养Jurkat细胞经流式细胞术测定有7.9%发生凋亡。该装置提供了一种确定何时诱导细胞凋亡、维持细胞活力以进行长期分析以及在细胞暴露于影响凋亡的试剂时实时观察细胞的方法。未来,该装置将成为研究细胞凋亡时间动态的宝贵工具。