Paguirigan Amy L, Puccinelli John P, Su Xiaojing, Beebe David J
Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin-Madison, Madison, Wisconsin 53726, USA.
Assay Drug Dev Technol. 2010 Oct;8(5):591-601. doi: 10.1089/adt.2010.0274. Epub 2010 Jul 26.
Microfluidic methods for cellular studies can significantly reduce costs due to reduced reagent and biological specimen requirements compared with many traditional culture techniques. However, current types of readouts are limited and this lack of suitable readouts for microfluidic cultures has significantly hindered the application of microfluidics for cell-based assays. The In-Cell Western (ICW) technique uses quantitative immunocytochemistry and a laser scanner to provide an in situ measure of protein quantities in cells grown in microfluidic channels of arbitrary geometries. The use of ICWs in microfluidic channels was validated by a detailed comparison with current macroscale methods and shown to have excellent correlation. Transforming growth factor-β-induced epithelial-to-mesenchymal transition of an epithelial cell line was used as an example for further validation of the technique as a readout for soluble-factor-based assays performed in high-throughput microfluidic channels. The use of passive pumping for sample delivery and laser scanning for analysis opens the door to high-throughput quantitative microfluidic cell-based assays that integrate seamlessly with existing high-throughput infrastructure.
与许多传统培养技术相比，用于细胞研究的微流控方法由于试剂和生物样本需求减少，可显著降低成本。然而，目前的读出方式有限，缺乏适用于微流控培养的读出方式严重阻碍了微流控技术在基于细胞的分析中的应用。细胞内蛋白质免疫印迹（In-Cell Western，ICW）技术利用定量免疫细胞化学和激光扫描仪，对在任意几何形状的微流控通道中生长的细胞内蛋白质数量进行原位测量。通过与当前宏观方法的详细比较，验证了ICW在微流控通道中的应用，并显示出极好的相关性。以转化生长因子-β诱导上皮细胞系发生上皮-间质转化为例，进一步验证了该技术作为高通量微流控通道中基于可溶性因子分析的读出方式的有效性。采用被动泵输送样品和激光扫描进行分析，为高通量定量微流控细胞分析打开了大门，该分析可与现有的高通量基础设施无缝集成。
