Xanthene dyes that emit to the red of fluorescein

• US 5,066,580 A
• Filed: 02/16/1990
• Issued: 11/19/1991
• Est. Priority Date: 08/31/1988
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Lee, Linda
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Stall, Jacintha M.
• Application Number:
  US07/481,010
• Time in Patent Office: 641 Days
• Field of Search: 435/7, 435/29, 435/34, 436/172, 436/800, 436/805, 436/7.24, 436/7.5, 436/19, 436/63, 424/3
• US Class Current: 435/7.21
• CPC Class Codes: C09B 11/28 Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

Novel xanthene dyes are disclosed which excite in the range of 450-650 nm and which will emit to the red of fluoroscein. These dyes may be coupled to tagging agents, such as monoclonal antibodies, and used to detect cells in a sample.

Claims

1. A method for staining cells in a sample which comprises incubating said with a tagging agent which specifically binds to said cells under conditions sufficient to allow said cells to bind with said tagging agent, said tagging agent being coupled to a fluorescent compound selected from the group consisting of ##STR3## wherein R is CO₂ H or SO₃ H, and X is H or Br when R is CO₂ H and X is H when R is SO₃ H;

##STR4##

2. The method of claim 1 wherein the tapping agent is in antibody.

3. The method of claim 2 wherein the antibody is a monoclonal antibody.

4. The method of claim 1 wherein the tagging agent is coupled to the compound via a biotin-avidin complex.

5. The method of claim 1 wherein the cells are derived from body tissue or body fluids.

6. The method of claim 1 wherein the compound is ##STR5##

7. The method of claim 1 wherein the compound is ##STR6##

8. A method for identifying cells in a sample which comprises:

(a) incubating said cells with a tagging agent which specifically binds to said cells under conditions sufficient to allow said cells to bind with said tagging agent, said tagging agent being coupled to a fluorescent compound selected from the group consisting of of the formula ##STR7## wherein R is CO₂ H or SO₃ H, and X is H or Br when R is CO₂ H and X is H when R is SO₃ H;

##STR8## (b) detecting fluorescence emitted by said bound tagging agent as a measure of the presence of said cells.

9. The method of claim 8 wherein the tagging agent is an antibody.

10. The method of claim 9 wherein the antibody is a monoclonal antibody.

11. The method of claim 8 in which the tagging agent is coupled to the compound via a biotin-avidin complex.

12. The method of claim 8 wherein the cells are derived from body tissue or body fluids.

13. The method of claim 8 wherein fluorescence is detected using a flow cytometer.

14. The method of claim 8 wherein the compound is ##STR9##

15. The method of claim 8 wherein the compound is ##STR10##

16. A method for discriminating between live cells and dead cells in a sample which comprises:

(a) incubating said cells with a fluorescent compound selected from the group consisting of ##STR11## wherein R is CO₂ H or SO₃ H, and X is H or Br when R is CO₂ H and X is H when R is SO₃ H;

##STR12## under conditions sufficient to allow uptake of said compound by said live cells; and

(b) detecting fluorescence emitted by said compound as a measure of the presence of said live cells.

17. The method of claim 16 wherein the compound is ##STR13##

18. The method of claim 16 wherein the compound is ##STR14##

19. A method for detecting an esterase activity in a sample of cells comprising

a) incubating said cells with a fluorescent compound selected from the group consisting of ##STR15## wherein R is CO₂ H or SO₃ H, and X is H or Br when R is CO₂ H and X is H when R is SO₃ H;

##STR16## under conditions sufficient to allow uptake of said compound by said cells and cleavage of said compound by said esterase activity

b) detecting fluorescence emitted by said cleaved compound as a measure of said esterase activity.

20. The method of claim 19 wherein the compound is ##STR17##

21. The method of claim 19 wherein the compound is ##STR18##