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首页> 外文期刊>Journal of Microbiological Methods >A pipeline for developing and testing staining protocols for flow cytometry, demonstrated with SYBR Green I and propidium iodide viability staining
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A pipeline for developing and testing staining protocols for flow cytometry, demonstrated with SYBR Green I and propidium iodide viability staining

机译：用于开发和测试流式细胞术染色方案的管道，通过SYBR Green I和碘化丙啶活力染色进行了演示

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The increasing use of flow cytometry (FCM) for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. Viability assessment with FCM is in this context of particular interest because incorrect staining could severely affect the outcome/interpretation of the results. Here we propose a pipeline for the development and optimization of staining protocols for environmental samples, demonstrated with the common viability dye combination of SYBR Green I (SG) and propidium iodide (PI). Optimization steps included the assessment of dye solvents, determination of suitable PI concentration, and determining the optimal staining temperature and staining time. We demonstrated that di methyl sulfoxide (DMSO) could impair membrane integrity, when used for SGPI stock solution preparation, and TRIS buffer was chosen as an alternative. Moreover we selected 6 mu M as optimal PI final concentration: less than 3 mu M resulted in incomplete staining of damaged cells, while concentrations higher that 12 mu M resulted in false Pl-positive staining of intact cells. Low temperatures (25 degrees C) resulted in a slow reaction and did not enable the staining of all bacteria, while high temperatures (44 degrees C) caused damage to cells'and false PI-positive results. Hence, 35 degrees C was selected as optimal staining temperature. We further showed that a minimum of 15 min were necessary to obtain stable staining results. Moreover, we showed that addition of EDTA resulted in 1-39% more Pl-positive results compared to an EDTA-free sample, and argue that insufficient evidence currently exist in favor of adding EDTA to all samples in general. Altogether, the data clearly shows the need to be careful, precise and reproducible when staining cells for flow cytometric analyses, and the need to assess and optimize staining protocols with both viable and non-viable bacteria. (C) 2016 Elsevier B.V. All rights reserved.
机译：流式细胞术（FCM）在环境样品分析中的使用日益增多，导致了各种各样的染色方案，但结果却有所不同，可比性也受到限制。在这种情况下，使用FCM进行生存力评估尤为重要，因为不正确的染色会严重影响结果/结果的解释。在这里，我们提出了开发和优化环境样品染色方案的管道，并通过SYBR Green I（SG）和碘化丙锭（PI）的常见活性染料组合进行了证明。优化步骤包括评估染料溶剂，确定合适的PI浓度以及确定最佳染色温度和染色时间。我们证明了，二甲基亚砜（DMSO）在用于SGPI储备溶液制备时会损害膜的完整性，因此选择了TRIS缓冲液作为替代品。此外，我们选择6μM作为最佳PI最终浓度：小于3μM导致受损细胞染色不完全，而高于12μM的浓度导致完整细胞假Pl阳性染色。低温（25摄氏度）导致反应缓慢，无法使所有细菌染色，而高温（44摄氏度）则导致细胞受损和假PI阳性结果。因此，选择35℃作为最佳染色温度。我们进一步表明，至少需要15分钟才能获得稳定的染色结果。此外，我们表明，与不含EDTA的样品相比，添加EDTA导致的Pl阳性结果多1-39％，并指出目前没有足够的证据支持将EDTA总体上添加到所有样品中。总之，数据清楚地表明，在对细胞进行流式细胞术分析时需要小心，准确和可重现，并且需要评估和优化活细菌和非活细菌的染色方案。 （C）2016 Elsevier B.V.保留所有权利。

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