White William N, Sethu Palaniappan
Department of Mechanical Engineering, University of Louisville, Louisville, KY, USA.
J Vis Exp. 2009 Dec 31(34):1656. doi: 10.3791/1656.
In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.
在本报告中，详细介绍了一种自动化微流控血液裂解装置的方案。循环有核细胞（CNC），包括白细胞和内皮细胞，为了解个体免疫状况的最新情况提供了一个理想的平台。该微流控方案能够富集CNC，且不会因用户操作步骤导致选择性细胞损失和样品制备差异。简而言之，该方案包括装置制造、样品采集、装置设置以及使血液流经微流控腔室。在装置内，全血在一个50微米×150微米的微流控通道中与去离子水快速混合约10秒。在此时间段内，红细胞因低渗条件而裂解。通道底部的人字形结构确保了充分混合以及细胞暴露于恒定环境。剩余细胞在装置出口处恢复到等渗条件，用2%多聚甲醛固定，离心以从CNC中分离出红细胞碎片，然后悬浮在流动缓冲液中用于流式细胞术染色和分析。结果显示流式细胞术散射图清晰，CNC群体得以保留。该装置和方案的意义在于通过分析CNC群体来研究和理解疾病发病机制。因此，证明了微流控方案的自动化、有效性和简易性。
