Question 1

这些染料如何与DNA结合？

Accepted Answer

SYTO核酸染料的结合方式尚不清楚。但是，SYTO以及相关的核酸染料具有以下结合特性：

1.它们与一些溶剂结合（通过对盐、二价阳离子的敏感性，特别是SDS），因此，它们可能结合到DNA沟槽处。
2.所有SYTO染料都表现出一些碱基选择性，因此，它们可能结合到DNA小沟处。
3.通过乙醇沉淀可从核酸中去除SYTO染料；溴化乙锭和其他嵌入剂不具有此特性。同样，丁醇和氯仿提取不能从核酸中去除SYTO染料，但能够去除溴化乙锭。
4.SYTO 结合不受非离子去垢剂的影响。
5.SYTO染料不会被BrdU淬灭，因此，SYTO染料与核酸的结合方式不同于Hoechst 33342和DAPI（4',6-二脒基-2-苯基吲哚）。

SYBR Green I对移码指示菌几乎没有致突变性，证明其不大可能是强嵌入剂。

Question 2

alamarBlue试剂和PrestoBlue试剂与刃天青和C12-刃天青有什么不同？

Accepted Answer

alamarBlue试剂和PrestoBlue试剂含有的刃天青包含在一种专有的稳定配方中，能够提供了一种便捷的“混合、接种和读数”实验方案。PrestoBlue试剂是alamarBlue试剂的改进配方允许更快速的染色（典型的10分钟相比于1-4小时获得相似的信号和灵敏度）。C12-刃天青是刃天青的衍生物，其拥有良好的细胞滞留性，因此适用于伴有活性指示剂和其他生物标记的流式细胞分析仪多重检测分析。

Question 3

我如何为LIVE/DEAD细胞活力实验准备死细胞对照？

Accepted Answer

有两种简单的方法。一种是通过60°C放置20分钟热灭活细胞。第二种是将细胞放入70%乙醇。乙醇固定的细胞可以在冰箱中无限期储存直到使用，可达好几年。

1.离心细胞、使细胞沉淀并去除上清
2.固定细胞：在15mL含有细胞团的试管中加入10 mL70%冰乙醇，先开始逐滴加入后震荡，混合均匀。
3.在冰箱中储存直到使用。
4.快使用时，水洗两次且在选择的缓冲液中重悬。

Question 4

How do SYTO dyes bind to DNA?

Accepted Answer

The binding mode of SYTO nucleic acid stains is unknown. However, the behavior of these and related nucleic acid dyes suggests the following binding properties:

1.They appear to contact the solvent (suggested by sensitivity to salt, divalent cations, and in particular, SDS) and thus are likely to have contacts in the grooves.
2.All SYTO dyes appear to show some base selectivity and are thus likely to have minor groove contacts.
3.They can be removed from nucleic acid via ethanol precipitation; this characteristic is not shared by ethidium bromide and other intercalators. Likewise, the dyes are not removed from nucleic acid via butanol or chloroform extraction. These extraction methods do remove ethidium bromide from nucleic acid. 4. SYTO binding is not affected by nonionic detergents.
5. SYTO dyes are not quenched by BrdU, so they do not bind nucleic acids in precisely the same way as Hoechst 33342 and DAPI ((4′,6-diamidino-2-phenylindole).

SYBR Green I has shown little mutagenicity on frameshift indicator strains, indicating that it isn't likely to strongly intercalate.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Question 5

How do alamarBlue reagent and PrestoBlue reagent differ from resazurin and C12-resazurin?

Accepted Answer

alamarBlue reagent and PrestoBlue reagent contain resazurin in a proprietary stabilizing formulation that allows for a convenient “mix, incubate, and read” protocol. PrestoBlue reagent is an improvement in the formulation of alamarBlue reagent that allows for much faster staining (typically 10 minutes vs. 1-4 hours to obtain a similar signal and sensitivity). C12-resazurin is a derivative of resazurin that has better cellular retention and thus allows for analysis on a flow cytometer and multiplexing with viability indicators and other biomarkers.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Question 6

How do I prepare dead cell controls for LIVE/DEAD cell viability assays?

Accepted Answer

There are two easy options. One is to heat-inactivate the cells by placing at 60 degrees C for 20 minutes. The second is to subject the cells to 70% ethanol. Alcohol-fixed cells can be stored indefinitely in the freezer until use, potentially up to several years.

Centrifuge cells, pellet, and remove supernatant.
Fix cells: Add 10 mL ice cold 70% ETOH to a 15 mL tube containing the cell pellet, adding dropwise at first while vortexing, mix well.
Store in freezer until use.
When ready to use, wash twice and resuspend in buffer of choice.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

LIVE/DEAD™ Cell Vitality Assay Kit, C12 Resazurin/SYTOX™ Green - FAQs