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DNA Dyes in Flow Cytometry and Microscopy

As your introductory science classes taught you, DNA is the blueprint for life. And, it tends to be an important focal point in many researchers' work. In this blog, we'll go over how DNA-binding dyes work and how they can be used in flow cytometry and microscopy applications.

Why are we looking for DNA?
DNA can provide lots of insight into the inner workings of a cell. In flow cytometry, we can use DNA-binding dyes to help assess the cell's health. If a dye is not cell-permeant (more on this later), it's excluded by a healthy and intact cell membrane. If that membrane is damaged or the cell is going through apoptosis, the dye can then finds its way to the nucleus and stain DNA. While some people will rely solely on forward and side scatter profiles to assess viability, this is not always reliable and may not include cells in the early stages of apoptosis that have not shrunken down as they die and become debris.

Versatile Helix NP™ Dyes

C57BL/6 mouse thymus cells were fixed using 70% chilled ethanol. The cells were incubated for one hour at -20°C, washed, then stained with Helix NP™ NIR at 5 μM. HeLa cells were fixed, permeabilized, and blocked. Then the cells were intracellularly stained with Alexa Fluor® 488 anti-Cytokeratin (pan reactive)(green) antibody followed by Helix NP™ NIR (red)
DNA dyes can even show you what stage of the cell cycle the cell is currently in. Due to the stoichiometric binding of nucleic acid stains, cells in the G2 or M phase have double the DNA content of a normal cell before dividing and thus, stain with much higher levels of Helix NP™ NIR and have a higher mean fluorescence intensity. DRAQ5™ and CytoPhase™ Violet can be used in a similar manner. In microscopy, the applications for DNA dyes are a little different. DNA dyes are typically used to counterstain cells or tissues. This gives better contextual and localization clues to proteins you've stained with antibodies.

Traits of DNA Dyes

Some of the important traits to understand about your DNA dye include permeability and their mechanism of action. Refer to our handy chart to keep track of these characteristics.
DNA Dye Equivalent Channel/Fluor Cell Permeable? Binding Mechanism
Helix NP™ Blue Sytox® Blue, Coumarin, BV421™ No Single intercalator
Helix NP™ Green Sytox® Green, FITC No Bis-intercalator
Helix NP™ NIR To-Pro-3®, Alexa Fluor® 647, APC No Single intercalator
CytoPhase™ Violet Hoechst 33258, Coumarin, BV421™ Yes Minor groove
DRAQ5™ Alexa Fluor® 647, APC Yes Single intercalator
DRAQ7 Alexa Fluor® 700, APC/Cyanine7 No Single intercalator
7-AAD PE/Cyanine5, PE/Dazzle™ 594 No Single intercalator
DAPI Coumarin, BV421™ Semi Minor groove

Permeability
Permeability is a simple enough concept: can the dye get into the cell to bind DNA if the membrane is intact? Permeability will define the type of assay your dye is useful for. If it's cell-permeant, it's less suitable for a viability assay since it will get into every cell regardless of its health status. Instead, it will work better to give you an idea of a total cell count or cell cycle status. If a dye is not cell-permeant, then it can be helpful in assessing viability. For our Helix NP™ dyes, NP indicates they're "Not cell-Permeant" so they're helpful assessors of cell health. Similarly, DRAQ7™ and Propidium Iodide are also excluded by healthy cells with intact membranes. Other dyes like DAPI and 7-AAD are classically considered not to be cell-permeant. However, they are actually semi-permeant, meaning at high concentrations, they'll get through a live cell's membrane and stain, but to a lesser degree than a dead cell would.

Mechanisms of Action

It can also be helpful to understand how these dyes are binding to DNA. Different interactions with DNA have different affinities. The higher the affinity, the tighter the binding and the more easily you can resolve DNA and see more than just a haze around the nucleus. We'll be focusing on dyes that bind the minor groove and intercalators. The minor groove is the narrower of the two grooves formed by the double helix structure of DNA. Minor groove binders generally have a lower affinity than intercalating dyes. Minor groove binders may be a little less flexible as they must follow the groove as it twists around the axis, coming into contact with the edges of base pairs. Binding typically occurs through non-covalent means (i.e., hydrogen bonding of the probe to base pairs).

 
DNA intercalators actually insert or "intercalate" non-covalently between two sets of adjacent base pairs, causing them to separate and create a pocket for the dye. A part of dye's structure, such as a hydrophobic aromatic ring that bears resemblance to a ring of bases in DNA, can insert itself between sets of base pairs. This can actually cause the DNA to become distorted. Bis-intercalators contain two intercalating moieties connected by a linker that interacts with a groove. As such, they have an even higher affinity than single intercalators and have even been used to image single strands of DNA. Minor groove binders and intercalators can increase in fluorescence due to conformation changes upon binding DNA. It is important to notice that most DNA-binding dyes can be dislodged if the samples are fixed or permeabilized. This is particularly important in scenarios where organic solvents with DNA-denaturing properties are used. For example, methanol is commonly used to help solubilize paraformaldehyde in certain fixation solutions. This can lead to false positives and negatives, making it harder to interpret your data. Alternatively, you can analyze viability with Zombie Dyes, which bind amines on proteins and are more well-suited for fixation and permeabilization conditions.
Hopefully, now you have a better understanding of how DNA-binding dyes work and which one you should choose for your applications. If you want to learn more about DNA dyes, other chemical probes, and their applications, check out our Cell Health and Proliferation Webpage. And if you still have questions, contact our tech support group.

Contributed by Ken Lau, PhD.
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