Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods
Inventors
Montange, Rebecca K. • Steaffens, Jeffrey W.
Assignees
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Publication Number
US-11709116-B2
Publication Date
2023-07-25
Expiration Date
Abstract
A kit and method for flow cytometry include a liquid dye concentrate for fluorescent staining of virus-size particles with a plurality of fluorogenic dyes in a liquid medium. The liquid dye concentrate includes a plurality of fluorogenic dyes and one or both of (i) the liquid medium comprising a liquid mixture including water and liquid phase organic material and (ii) disaccharide dissolved in the liquid medium.
Core Innovation
The invention addresses inconsistent flow-cytometry staining of unassociated virus-size particles. The disclosure attributes performance problems to dye aggregation, pi-stacking, and reconstitution variability in aqueous media from dry powders, and focuses on multi-fluorogenic-dye formulations and associated fluorescent stain products and kits to improve staining performance and reduce variability in detection of particle-bound fluorescence signatures.
The disclosure provides a kit including a liquid dye concentrate with a plurality of different fluorogenic dyes dissolved in a liquid medium, where each fluorogenic dye has a different fluorescent emission signature for detection when bound to a particle. The kit further includes an aqueous sample dilution liquid contained in a sealed second container for preparing aqueous diluted fluid samples containing biological material for flow cytometry evaluation.
The liquid dye concentrate and fluorescent stain product are formulated with a liquid phase organic material and/or disaccharide in specified concentrations. In the stained fluid sample concepts, multiple fluorogenic dyes are used for direct fluorescent staining of aqueous fluid samples, with the dyes having different fluorescent emission signatures and containing at least one aromatic group susceptible to pi stacking, to separate true particle fluorescence from free dye aggregates and support improved repeatability and shelf life.
Claims Coverage
The partial content includes five independent claims. Across these claims, there are inventive features centered on multi-fluorogenic-dye formulations packaged for flow cytometry, defined liquid medium composition using liquid phase organic material and/or disaccharide, and a flow cytometry evaluation method that omits reconstitution from dry form.
Sealed multi-fluorogenic dye concentrate and aqueous dilution liquid in a kit
A kit for preparing fluorescently stained fluid samples containing biological material for flow cytometry evaluation for particles, the kit comprising a liquid dye concentrate in a sealed first container having a plurality of different fluorogenic dyes dissolved in a liquid medium with different fluorescent emission signatures when bound to a particle, and an aqueous sample dilution liquid in a sealed second container for preparing aqueous diluted fluid samples.
Liquid medium comprising liquid phase organic material or disaccharide within defined wt% ranges
The kit wherein the liquid dye concentrate comprises the liquid medium comprising liquid phase organic material at a concentration from 10 weight percent to 50 weight percent, and/or disaccharide dissolved in the liquid medium at a concentration from 1 weight percent to 45 weight percent of the liquid dye concentrate, including combinations thereof.
Dye concentrate liquid medium including DMSO
The kit wherein the liquid dye concentrate comprises a liquid medium comprising liquid phase organic material at a concentration from 10 weight percent to 50 weight percent, or disaccharide dissolved in the liquid medium at a concentration from 1 weight percent to 45 weight percent of the liquid dye concentrate, and wherein the liquid phase organic material comprises dimethyl sulfoxide (DMSO).
Fluorescent stain product with multiple fluorogenic dyes in a sealed container
A fluorescent stain product comprising a liquid dye concentrate having a plurality of different fluorogenic dyes dissolved in a liquid medium where each different fluorogenic dye has a different fluorescent emission signature when bound to a particle, and a sealed container containing the liquid dye concentrate.
Flow cytometry evaluation method using liquid dye concentrate in the absence of reconstituting from dry form
A method for flow cytometry evaluation of biological material for particles comprising providing an aqueous preliminary fluid sample, providing a liquid dye concentrate with a plurality of different fluorogenic dyes dissolved in a liquid medium and wherein providing the liquid dye concentrate is in the absence of reconstituting the fluorogenic dyes from a dry form into the liquid medium, preparing an aqueous stained fluid sample by staining the preliminary fluid sample with the liquid dye concentrate, and subjecting the stained fluid sample to flow cytometry to detect and count occurrences of the particles stained with at least one of the fluorogenic dyes.
The claim set centers on kits and products that provide multi-fluorogenic dye formulations with distinct fluorescent emission signatures in sealed formats for flow cytometry evaluation, with liquid dye concentrate media defined by liquid phase organic material and/or disaccharide within specified weight-percent ranges. A further inventive emphasis is a flow cytometry evaluation method that stains using a liquid dye concentrate provided in the absence of reconstituting fluorogenic dyes from a dry form.
Stated Advantages
Improves repeatability.
Improves shelf life.
Helps distinguish true particle fluorescence from free dye aggregates.
Lowers coefficient of variation (c.v.).
Reduces blank particle counts.
Reduces dye aggregation and pi-stacking in aqueous media.
Documented Applications
Direct fluorescent staining of aqueous fluid samples containing biological materials with multiple fluorogenic dyes for flow cytometry evaluation of stained fluid samples for particles.
Flow cytometry evaluation of biological material for particles by detecting and counting occurrences of particles stained with at least one of the fluorogenic dyes.
Preparing fluorescently stained fluid samples containing biological material for flow cytometry evaluation for particles using a kit comprising a sealed liquid dye concentrate and a sealed aqueous sample dilution liquid.
Using the disclosed stained fluid sample for flow cytometry evaluation for particles dispersed in an aqueous liquid medium.
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