Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
2001-04-26
2002-06-11
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S063000, C436S164000, C436S166000, C436S172000, C435S029000, C252S408100
Reexamination Certificate
active
06403378
ABSTRACT:
BRIEF DESCRIPTION OF THE INVENTION
This invention relates generally to a cell viability assay reagent used for staining cells in a cell suspension so that all non-viable cells are stained to fluoresce at a first wavelength responsive to light at one wavelength, and all viable and non-viable cells are stained to fluoresce at a second wavelength responsive to excitation by light of a lower wavelength.
BACKGROUND OF THE INVENTION
Two dyes are generally used to stain cells in a suspension for viability analysis. One dye consists of a membrane permeant DNA dye that labels all intact cells in a suspension, whereby they emit light at one wavelength. A non-permeant DNA dye labels all dead cells.
In one method of analysis, the cells in the cell suspensions are stained and a traditional hemacytometer is used to differentiate the cells. Another analysis system utilizes dual-color fluorescence in combination with forward light scatter to determine the concentration of nucleated cells and cell viability. Cells are analyzed by providing relative movement between the sample suspension containing the cells and an excitation light beam, whereby labeled cells pass through the light beam and emit light at a wavelength characteristic of the permeant and non-permeant dye. The detection system includes filters and detectors which detect the light emitted at the two wavelengths. The cells also scatter light, whereby all particles in the sample suspension are detected. Once a cell has been detected on the permeant dye channel, the light scatter profile is evaluated to assure that the cell is of sufficient size to be an intact cell and not simply a free nucleus or other cell fragment. The second dye permeates all cells with damaged or “leaky” membranes. The dye emits fluorescent light at a different wavelength range than that of the cells stained with permeant dye. In this manner all cells are detected by detecting the light emitted by the second dye at one wavelength, and non-viable cells are detected by detecting light emitted by the permeant dye at the other wavelength. Thus, an absolute count of cells and percent viability can be obtained from the data.
To obtain reliable results for different cell concentrations, using a two-dye method it is necessary to carefully control the amount of each of the dyes used to stain or tag the cells. This is a time-consuming procedure and may lead to variability in results obtained.
SUMMARY OF THE INVENTION
The present invention is directed to a cell viability reagent which uses C
25
H
30
ClN
3
O
4
obtained from Exciton Laboratories (herein LDS) and propidium iodide obtained from Sigma Chemical Company (Cat. #P4120, herein PI) to stain the cells so they emit fluorescent light having a peak value at 580 nm and 675 nm, respectively, responsive to excitation by light at 532 nm. The dye is used in a cell analysis system designed to detect the fluorescent light at the two wavelengths and provide an output indicative of cells fluorescing at one or both wavelengths.
REFERENCES:
patent: 3586859 (1971-06-01), Katz et al.
patent: 5047321 (1991-09-01), Loken et al.
patent: 5057413 (1991-10-01), Terstappen et al.
patent: 5185450 (1993-02-01), Owen
patent: 5314805 (1994-05-01), Haugland et al.
patent: 5879900 (1999-03-01), Kim et al.
Frey, Tom. Detection of Bromodeoxyuridine Incorporation by Alteration of the Flourescence Emission from Nicleic Acid Binding Dyes Using Only an Argon Ion Laser. Cytometry, vol. 17, pp. 310-318, 1994.
Phi-Wilson Janette T.
Rackham Don F.
Sheehan Kevin M.
Flehr Hohbach Test Albritton & Herbert LLP
Guava Technologies, Inc.
Wallenhorst Maureen M.
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