Beal Jacob, Telmer Cheryl A, Vignoni Alejandro, Boada Yadira, Baldwin Geoff S, Hallett Liam, Lee Taeyang, Selvarajah Vinoo, Billerbeck Sonja, Brown Bradley, Cai Guo-Nan, Cai Liang, Eisenstein Edward, Kiga Daisuke, Ross David, Alperovich Nina, Sprent Noah, Thompson Jaclyn, Young Eric M, Endy Drew, Haddock-Angelli Traci
Intelligent Software and Systems, Raytheon BBN Technologies, 10 Moulton Street, Cambridge 02138, MA, USA.
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh 15213, PA, USA.
Synth Biol (Oxf). 2022 Aug 6;7(1):ysac010. doi: 10.1093/synbio/ysac010. eCollection 2022.
Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.
酶标仪常用于测量细胞生长和荧光，但酶标仪数据的实用性和可重复性受到其通常以任意或相对单位报告这一事实的限制。我们之前已经建立了一个稳健的系列稀释方案，用于将酶标仪测量的吸光度校准为估计的细菌细胞数量，以及将细菌细胞中表达的蛋白质的绿色荧光校准为等效荧光素分子。我们现在将这些方案扩展到将红色荧光校准为磺罗丹明-101荧光染料，将蓝色荧光校准为级联蓝。通过一项实验室间研究评估校准效果，我们发现这些校准物确实能提供与先前校准物相当的精度，并且它们能够实现对细菌细胞中表达的蛋白质的红色和蓝色荧光测量的有效跨实验室比较。
