Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria.
Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria.
J Biotechnol. 2018 Feb 20;268:40-52. doi: 10.1016/j.jbiotec.2018.01.008. Epub 2018 Jan 20.
Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg and Ca, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry.
为了基于荧光信号研究细菌生理学和基因表达参数，在流式细胞术分析中，缓冲液对于稀释细菌培养物至关重要。我们使用大肠杆菌 K-12 菌株 MG1655 中的各种组成型表达的荧光蛋白，发现将其稀释到常用的流式细胞术缓冲液磷酸盐缓冲盐水（PBS）和另外两种缓冲液 Tris-HCl 和 M9 盐中后，荧光水平会出现强烈的人为变化。这些变化在稀释后很快出现，与膜通透性增加和细胞活力丧失有关。我们在几种不同的大肠杆菌菌株（K-12、C 和 W）中观察到了与缓冲液相关的效应，但在 E. coli B 中没有观察到，这可以部分解释为脂多糖（LPS）和外膜组成的差异。用负责外膜稳定性的二价阳离子（Mg 和 Ca）补充缓冲液，可以维持大肠杆菌的荧光信号、膜完整性和活力。因此，稳定细菌外膜对于使用流式细胞术进行荧光参数的精确和无偏测量至关重要。
