Automated High-Throughput Microchannel Assays for Cell Biology: Operational Optimization and Characterization
Automated High-Throughput Microchannel Assays for Cell Biology: Operational Optimization and Characterization
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DOI：
10.1016/j.jala.2009.10.002
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发表时间：
2010-02-01
期刊：
影响因子：
--
通讯作者：
Beebe, David J.
中科院分区：
文献类型：
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作者：
Puccinelli, John P.;Su, Xiaojing;Beebe, David J.
细胞培养中筛选生物读数的频率和通量不断增加。在此类测定中，细胞类型可能稀有并且试剂或化合物可能昂贵，通常导致条件和/或重复数量减少。正如之前所表明的，“无管”微流体技术提供了一种减轻这种负担的方法。此外，In-Cell Western (ICW) 最近已适应微流体培养，允许在微流体通道中进行免疫细胞化学的高通量分析。将无管微流控中的自动液体处理与 ICW 相结合，可提供快速、定量的高通量细胞筛选。在这里，我们使用三个参数验证该平台：操作稳健性（移液可靠性）、细胞接种一致性和细胞染色一致性（核和抗体）。液体处理与微流体的集成被发现具有超过 97% 的运行稳健性。发现每个微通道之间和每个微通道内的细胞接种一致性的标准偏差分别小于5%和6%。最后，通过优化液体处理步骤，发现所有通道的核染色和抗体染色均具有一致性。这些结果为使用自动化无管微流体执行大多数标准 ICW 测定奠定了基础。 （贾拉 2010；15:25-32）
Screening biological readouts in cell culture are increasing in frequency and throughput. In such assays, cell types may be rare and reagents or compounds may be expensive often resulting in a reduced number of conditions and/or replicates. "Tubeless" microfluidics offers a method to reduce this burden, as has been previously shown. In addition the In-Cell Western (ICW) has recently been adapted to microfluidic cultures allowing high throughput analysis of immunocytochemistry in microfluidic channels. Combining automated liquid handling in tubeless microfluidics with the ICW provides rapid and quantitative high throughput cell-based screens. Here, we validate this platform using three parameters: operational robustness (pipetting reliability), cell seeding consistency, and cell staining consistency (both nuclear and antibody). Integration of liquid handling with microfluidics was found to be more than 97% operationally robust. Cell seeding consistency between each microchannel and within each microchannel was found to be within a standard deviation of less than 5% and 6%, respectively. Finally, through optimization of liquid handling steps, uniformity among all the channels was found for both nuclear and antibody staining. These results lay the foundation to perform most standard ICW assays using automated tubeless microfluidics. (JALA 2010;15:25-32)