Fixable Viability Dyes for Stable Intracellular Cell Staining
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Solution Overview
Problem
Existing DNA binding dyes used for distinguishing live and dead cells in flow cytometry are not suitable for intracellular staining due to detachment after fixation and permeabilization, and NHS-ester dyes are unstable and require special handling.
Innovation Solution
Use of labelling compounds with a maleimide moiety linked to a fluorescent label that binds to free thiol groups on cell surface and intracellular proteins of dead cells, allowing differentiation by increased labelling intensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA binding dyes are used to distinguish live and dead cells, then cell viability can be detected, but the dyes detach after fixation and permeabilization, making them unsuitable for intracellular staining
Solution Approach 1:
The patent changes the chemical binding parameter from non-covalent binding (DNA binding dyes) to covalent binding (maleimide-thiol conjugation). This chemical parameter change ensures the dye remains bound to cell surface proteins even after fixation and permeabilization, resolving the stability issue while maintaining viability detection capability
Solution Approach 2:
The patent uses cell surface proteins with free thiol groups as an intermediary binding target. The maleimide moiety forms a stable covalent bond with these thiol groups, providing a stable anchor point for the fluorescent dye on the cell surface that persists through fixation and permeabilization steps
2Adaptability or versatility
If NHS-ester dyes are used to stain cell surface proteins, then intracellular staining can be performed, but the dyes are unstable and require special handling at -20°C
Solution Approach 1:
The patent changes the solvent parameter from DMSO (required for NHS-ester dyes) to aqueous buffer. The maleimide-containing dye is stable in aqueous solutions at room temperature, eliminating the need for special cold storage and DMSO handling while maintaining the ability to perform intracellular staining
Solution Approach 2:
The patent adopts a simpler, more stable dye molecule that can be stored and handled under ordinary conditions rather than requiring complex cold chain logistics. The dye is designed to be stable enough for routine laboratory handling but functional enough for the specific application
3Reliability
If NHS-ester dyes are used for viability staining, then live and dead cells can be differentiated, but the dyes must be aliquoted and re-suspended in DMSO just prior to use
Solution Approach 1:
The patent performs the conjugation of the fluorescent label to the maleimide moiety during the dye synthesis process, creating a pre-conjugated stable compound. This preliminary action eliminates the need for laboratory personnel to perform the conjugation step just before use, simplifying the workflow while maintaining reliability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Accurately distinguishes dead cells from live cells with at least 10 times greater labelling intensity, suitable for high-throughput assays like flow cytometry, and stable at room temperature without special handling.
Implementation Method 1
The labelling compound binds free thiol groups on cell surface proteins on dead cells and live cells and preferentially crosses cell membranes of dead cells and binds free thiol groups on intracellular proteins in the dead cells
Implementation Method 2
The label is a fluorescent label... excitation/emission wavelength of 402 nm/425 nm, a molecular weight of 1186.27 g/mol and an excitation/emission wavelength of 638 nm/652 nm, or a molecular weight of 1647.66 g/mol and an excitation/emission wavelength of 852 nm/877 nm
Data Source
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AI summary
The present invention provides compositions and methods useful for detecting live cells and dead cells in a cell sample. The methods rely on labelling compounds comprising a maleimide moiety linked to a label.