Dual-fluorescence cell viability assay using ethidium homodimer and calcein AM

• US 5,314,805 A
• Filed: 10/28/1991
• Issued: 05/24/1994
• Est. Priority Date: 10/28/1991
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Moore, Patrick L., MacCoubrey, Ian C., Haugland, Richard P.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): GITOMER, RALPH J
• Application Number:
  US07/783,182
• Time in Patent Office: 939 Days
• Field of Search: 435/6, 435/29, 435/34, 435/968, 436/800
• US Class Current: 435/29
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• 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

Abstract

This invention relates to a method for simultaneously detecting live and dead cells using two fluorogenic reagents: calcein AM and ethidium homodimer. Live cells are distinguished by an intense uniform green fluorescence generated by the enzymatic hydrolysis of calcein AM: ##STR1## Dead or damaged cells are distinguished by a bright red fluorescence resulting from nucleic acids stained with ethidium homodimer: ##STR2## The assay is useful to determine cell viability and to monitor cytotoxicity agents or events.

Claims

1. A method for evaluating the viability of cells in a sample, comprising:

a) combining a sample containing cells with a concentration of calcein AM sufficient to yield detectable green fluorescence in any live cells present in said sample and a concentration of ethidium homodimer sufficient to yield detectable red fluorescence in any dead cells present in said sample;

b) viewing the sample with means for detecting the red and green fluorescence of cells.

2. A method, as claimed in claim 1, where said means for detecting fluorescence is fluorescent microscopy.

3. A method, as claimed in claim 1, where said means for detecting fluorescence is a multi-well fluorescent plate scanner.

4. A method, as claimed in claim 1, where said means for detecting fluorescence is flow cytometry.

5. A method, as claimed in claim 1, where said means for detecting fluorescence is fluorescence spectroscopy.

6. A method, as claimed in claim 1, where the concentration of calcein AM is greater than 0 and less than 10 μ

M.

7. A method, as claimed in claim 1, where the concentration of ethidium homodimer is greater than 0 and less than 10 μ

M.

8. A method, as claimed in claim 6, where the concentration of ethidium homodimer is greater than 0 and less than 10 μ

M.

9. A method, as claimed in claim 1, where the sample of cells has been exposed to a cytotoxic event.

10. A method, as claimed in claim 9, where the cytotoxic event is adding an organic chemical reagent or adding a biological agent or a temperature change or a barometric change or any combination thereof.

11. A method, as claimed in claim 10, where the cytotoxic event is adding a biological agent.

12. A method, as claimed in claim 10, where the cytotoxic event is a temperature change.

13. A method, as claimed in claim 10, where the cytotoxic event is a barometric change.

14. A method, as claimed in claim 10, where the cytotoxic event is adding an organic chemical reagent.

15. A method, as claimed in claim 9, further comprising sorting cells exhibiting red fluorescence from cells exhibiting green fluorescence.

16. A method for evaluating the viability of cells in a sample, comprising:

a) combining a sample containing cells with a concentration of calcein AM sufficient to yield detectable green fluorescence in live cells present in said sample, and a concentration of ethidium homodimer sufficient to stain nuclei in dead cells present in said sample with red fluorescence;

b) allowing sufficient time for both calcein AM and ethidium homodimer to yield optimal fluorescence;

c) exciting both calcein AM and ethidium homodimer at less than about 500 nm and greater than about 485 nm; and

d) viewing the sample with means for detecting the red and green fluorescence in the cells.

17. A method, as claimed in claim 16, where the cells are single cells.

18. A method, as claimed in claim 16, where the concentration of calcein AM is greater than 0.1 μ

M and less than 10 μ

M and the concentration of ethidium homodimer is greater than 0.1 μ

M and less than 10 μ

M.

19. A method, for analyzing the viability of single cells, comprising:

a) combining a sample containing said cells with a concentration of calcein AM sufficient to yield detectable green fluorescence in live cells present in said sample without staining dead cells present in said sample, where the concentration of calcein AM is between 0.1 μ

M and 10 μ

M, and a concentration of ethidium homodimer sufficient to stain nuclei in dead cells present in said sample with red fluorescence without staining live cells, where said concentration of ethidium homodimer is between 0.1 μ

M and 10 μ

M;

b) incubating the sample with both calcein AM and ethidium homodimer for 5 to 60 minutes;

c) exciting both calcein AM and ethidium homodimer at 485-500 nm; and

d) viewing the sample with means for detecting the red and green fluorescence in the cells.

20. A method as claimed in claim 19, further comprising sorting or counting the live cells or dead cells based on their fluorescence.