Methods, kits and stain compositions for flow cytometry evaluation of unassociated virus-size particles using multiple fluorogenic dyes
In flow cytometry evaluation for unassociated virus-size particles stained with multiple fluorogenic dyes, the fluid sample is stained with an aqueous diluted dye formulation including multiple fluorogenic dyes. Preparation of the aqueous diluted dye formulation includes first preparatory processing to prepare a concentrated dye formulation with the fluorogenic dyes provided in a dry powder mixture mixed with and dissolved into a first liquid medium comprising DMSO. After the first preparatory processing, aqueous liquid diluent is added to dilute the first liquid medium and while the fluorogenic dyes remain in solution the liquid medium is converted to an aqueous liquid medium, which may include DMSO as a minor molar component but at a significant concentration. The stained fluid sample may include dissolved disaccharide.
1. A fluorogenic stain composition for fluorescent staining of unassociated virus-size particles with multiple fluorogenic dyes for flow cytometry evaluation for the stained unassociated virus-size particles in a fluid sample, the fluorogenic stain composition comprising:
a plurality of different fluorogenic dyes each having a different fluorescent emission signature and each containing at least one aromatic group susceptible to pi stacking, the plurality of different fluorogenic dyes including at least a first fluorogenic dye with a first fluorescent emission signature and a second fluorogenic dye with a second fluorescent emission signature that is different than the first fluorescent emission signature; and
an aqueous liquid medium in which the plurality of different fluorogenic dyes are dissolved, the aqueous liquid medium comprising a disaccharide dissolved in the aqueous liquid medium.
2. The fluorogenic stain composition of claim 1 , comprising the disaccharide at a concentration of at least 1 weight percent.
3. The fluorogenic stain composition of claim 2 , comprising the disaccharide at a concentration of not greater than 45 weight percent.
4. The fluorogenic stain composition of claim 3 , comprising a weight-to-volume concentration of the first fluorogenic dye and a weight-to-volume concentration of the second fluorogenic dye each in a range of from 1 to 1000 micrograms per milliliter.
5. The fluorogenic stain composition of claim 3 , wherein the disaccharide comprises trehalose.
6. The fluorogenic stain composition of claim 3 , comprising one or more disaccharide materials, wherein the one or more disaccharide materials comprise trehalose, sucrose, lactose, lactulose, melibiose, melibiulose, cellobiose, nigerose, isomaltose, isomaltulose, maltulose, rutinose, β-laminaribiose, or maltose, or combinations thereof.
7. The fluorogenic stain composition of claim 1 , wherein the first fluorogenic dye is for nonspecific nucleic acid staining and the second fluorogenic dye is for nonspecific protein staining.
8. The fluorogenic stain composition of claim 7 , wherein each of the first fluorogenic dye and the second fluorogenic dye is a cyanine dye.
9. A method for flow cytometry evaluation of a fluid sample for unassociated virus-size particles stained with multiple fluorogenic dyes susceptible to pi stacking interactions in aqueous liquid, the method comprising:
preparing a stained fluid sample for flow cytometry evaluation, comprising mixing at least a portion of a fluorogenic stain composition with a sample of biological material to be evaluated by flow cytometry for presence of the unassociated virus-size particles;
after the preparing, subjecting the stained fluid sample to flow cytometry in a flow cytometer to detect and count occurrences of the unassociated virus-size particles stained with both a first fluorogenic dye and a second fluorogenic dye; and
wherein the fluorogenic stain composition comprises:
a plurality of different fluorogenic dyes each having a different fluorescent emission signature and each containing at least one aromatic group susceptible to pi stacking, the plurality of different fluorogenic dyes including at least the first fluorogenic dye with a first fluorescent emission signature and the second fluorogenic dye with a second fluorescent emission signature that is different than the first fluorescent emission signature, and wherein the first fluorogenic dye is for nonspecific nucleic acid staining and the second fluorogenic dye is for nonspecific protein staining; and
an aqueous liquid medium in which the plurality of different fluorogenic dyes are dissolved, the aqueous liquid medium comprising a disaccharide dissolved in the aqueous liquid medium.
10. The method of claim 9 , wherein the fluorogenic stain composition comprises the disaccharide at a concentration in a range of from 1 weight percent to 45 weight percent.
11. The method of claim 10 , wherein the first fluorogenic dye is for nonspecific nucleic acid staining and the second fluorogenic dye is for nonspecific protein staining.
12. The method of claim 11 , wherein the unassociated virus-size particles have a size in a range of from 10 nanometers to one micron.
13. The method of claim 12 , wherein the flow cytometry comprises:
hydrodynamically focusing a flow of the stained fluid sample with a sheath fluid; and
flowing the hydrodynamically focused stained fluid sample through a flow cell in which the flowing hydrodynamically focused stained fluid sample is subjected to excitation radiation to stimulate a fluorescent emission response from each of the first fluorogenic dye and the second fluorogenic dye on the unassociated virus-size particles stained with both the first fluorogenic dye and the second fluorogenic dye; and
separately detecting for each of the first fluorescent emission signature and the second fluorescent emission signature and time correlating detection of the first fluorescent emission signature and detection of the second fluorescent emission signature to determine a detection event indicative of an occurrence of a said unassociated virus-size particle stained with both the first fluorogenic dye and the second fluorogenic dye.
14. A method for flow cytometry evaluation of a stained fluid sample for unassociated virus-size particles stained with multiple fluorogenic dyes susceptible to pi stacking interactions in aqueous liquid, the method comprising:
subjecting the stained fluid sample to flow cytometry evaluation for the unassociated virus-size particles, wherein the stained fluid sample comprises:
biological material to be evaluated by flow cytometry for presence of the unassociated virus-size particles;
a plurality of different fluorogenic dyes each having a different fluorescent emission signature and each containing at least one aromatic group susceptible to pi stacking, the plurality of different fluorogenic dyes including at least a first fluorogenic dye with the first fluorescent emission signature and a second fluorogenic dye with a second fluorescent emission signature that is different than the first fluorescent emission signature; and
an aqueous liquid in which the biological material and fluorogenic dyes are dispersed;
wherein the flow cytometry evaluation comprises subjecting the stained fluid sample to flow cytometry in a flow cytometer to detect and count occurrences of the unassociated virus-size particles stained with both the first fluorogenic dye and the second fluorogenic dye; and
wherein the aqueous liquid of the stained fluid sample comprises dissolved disaccharide.
15. The method of claim 14 , wherein the stained fluid sample comprises a concentration of the dissolved disaccharide of at least 0.5 weight percent.
16. The method of claim 15 , wherein the stained fluid sample has a concentration of dissolved disaccharide of not greater than 15 weight percent.
17. The method of claim 16 , wherein the wherein the first fluorogenic dye is for nonspecific nucleic acid staining and the second fluorogenic dye is for nonspecific protein staining.
18. The method of claim 14 , wherein the unassociated virus-size particles have a size in a range of from 10 nanometers to one micron.
19. The method of claim 18 , wherein the flow cytometry comprises:
hydrodynamically focusing a flow of the stained fluid sample with a sheath fluid; and
flowing the hydrodynamically focused stained fluid sample through a flow cell in which the flowing hydrodynamically focused stained fluid sample is subjected to excitation radiation to stimulate a fluorescent emission response from each of the first fluorogenic dye and the second fluorogenic dye on the unassociated virus-size particles stained with both the first fluorogenic dye and the second fluorogenic dye; and
detecting for both the first fluorescent emission signature and the second fluorescent emission signature; and
wherein, the flowing comprises maintaining a flow rate of the hydrodynamically focused stained fluid sample through the flow cell in a range of from 300 nanoliters per minute to 6000 nanoliters per minute.
20. The method of claim 19 , wherein the flow cytometry comprises separately detecting for each of the first fluorescent emission signature and the second fluorescent emission signature and time correlating detection of the first fluorescent emission signature and detection of the second fluorescent emission signature to determine a detection event indicative of an occurrence of a said unassociated virus-size particle stained with both the first fluorogenic dye and the second fluorogenic dye.