# US 2021/0278331 A1 · Fixable viability dyes and their uses

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0278331 A1
- Document ID: US20210278331A1
- Status: Published application
- Kind: A1
- Application number: 17/256,523
- Filed: 2019-07-12
- Published: 2021-09-09
- 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/US20210278331A1

## 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 I:
wherein R is a label, or a label attached through a linker,
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.

### 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 extra cellular targets.

### Claim 5 (depends on 1)

The method of claim 1, wherein the label is a direct label.

### Claim 6 (depends on 5)

The method of claim 5, wherein the direct label is a fluorescent label.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (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 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13

A kit for distinguishing dead cells from live cells in a cell sample, the kit comprising
a container comprising a labelling compound of Formula I:
wherein R is a label, or a label attached through a linker,
one or more containers comprising reagents for fixing and permeabilizing cells in the sample, and
instructions on how to distinguish dead cells from live cells in a cell sample using the labelling compound

### Claim 14 (depends on 13)

The kit of claim 13, wherein the label is a direct label.

### Claim 15 (depends on 14)

The kit of claim 14, wherein the direct label is a fluorescent label.

### Claim 16 (depends on 13)

The kit of claim 13, wherein the labelling compound is in dry form.

### Claim 17 (depends on 13)

The kit of claim 13, which is suitable for distinguishing dead cells from live cells in a cell sample using a flow cytometer.

### Claim 18 (depends on 13)

The kit of claim 13, further comprising labelled binding agents for detecting intracellular and/or extra cellular targets in the cell sample.