# US 12,601,673 B2 · Fixable viability dyes and their uses

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- Number: US 12,601,673 B2
- Document ID: US12601673B2
- Status: Granted
- Kind: B2
- Application number: 17/256,523
- Filed: 2019-07-12
- Published: 2021-09-09
- Granted: 2026-04-14
- Earliest claimed: 2018-07-12
- Assignees: Beckman Coulter, Inc.
- Applicants: Beckman Coulter, Inc.
- Inventors: Tewfik Miloud; Shankar Pattabhiraman; James Tung
- Web page: https://patentgrep.com/patent/US12601673B2

## CPC

- G01N 15/14: Optical investigation techniques, e.g. flow cytometry
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/5094: {for blood cell populations (red blood cells G01N33/80)}
- G01N 33/582: {with fluorescent label}
- G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 1

A method for distinguishing dead cells from live cells in a cell sample, the method comprising:
contacting the cell sample with a labelling compound of formula:
wherein LABEL is a fluorescent label, wherein the labelling compound has
a molecular weight of 714.69 g/mol and an 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,
under conditions whereby 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; and
detecting the label in the cell sample, thereby distinguishing dead cells from live cells in the cell sample by detecting increased labelling of the dead cells, as compared to live cells, and as compared to a control cell sample stained with an NHS ester compound based dye.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method further comprises labelling intracellular targets in the cells after the step of contacting the cell sample with the labelling compound.

### Claim 3 (depends on 2)

The method of claim 2, wherein the step of labelling intracellular targets includes fixing and permeabilizing the cells.

### Claim 4 (depends on 2)

The method of claim 2, wherein the method further comprises labelling extracellular targets.

### Claim 5 (depends on 1)

The method of claim 1, wherein the conditions include a pH of between 6.5 and 7.5.

### Claim 6 (depends on 1)

The method of claim 1, wherein the labeling of dead cells is at least 20 times the labeling of live cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the step of contacting includes incubating the cell sample with the labelling compound for between 10 and 60 minutes.

### Claim 8 (depends on 1)

The method of claim 1, wherein the step of detecting the label in the cell sample is carried out using a flow cytometer.

### Claim 9 (depends on 1)

The method of claim 1, wherein the cell sample is a blood cell sample.

### Claim 10 (depends on 1)

The method of claim 1, wherein the cell sample is from a human.