Red fluorescent aldehyde dehydrogenase (ALDH) substrate

Patent metadata

Field	Value
Publication number	US-10156521-B2
Application number	US-201414767195-A
Country	US
Kind code	B2
Filing date	Feb 21, 2014
Priority date	Feb 21, 2013
Publication date	Dec 18, 2018
Grant date	Dec 18, 2018

Abstract

A detectable substrate for aldehyde dehydrogenase (ALDH) can be used for selecting cells that express ALDH. The detectable substrate can have a fluorescent moiety that has an excitation wavelength, an emission wavelength, or both, that does not overlap with the excitation wavelength, emission wavelength, or both, of green fluorescent protein.

First claim

That which is claimed:

1. A detectable substrate for aldehyde dehydrogenase (ALDH) comprising a compound having the following structure:

wherein the detectable substrate accumulates in a cell expressing ALDH in an amount sufficient to be detected by fluorescence.

2. A method for distinguishing ALDH-expressing cells in a mixed population of cells, the method comprising:
contacting the mixed population of cells with the detectable ALDH substrate of claim 1 ;
measuring a fluorescence of the mixed population of cells; and
identifying cells exhibiting an increased fluorescence relative to a fluorescence from the mixed population of cells before being contacted with the detectable ALDH substrate.

3. The method of claim 2 , wherein measuring the fluorescence is performed by fluorescence microscopy.

4. The method of claim 2 , wherein measuring the fluorescence is performed by fluorescence activated cell sorting (FACS).

5. The method of claim 2 , wherein at least a portion of the cells in the population express a fluorescent protein.

6. The method of claim 2 , wherein the detectable ALDH substrate has a peak emission wavelength that is longer than a peak emission wavelength of a fluorescent protein, if present, by about 30 nm or more.

7. The method of claim 2 , wherein the mixed population of cells is contacted with the detectable ALDH substrate in the presence of a multi-drug efflux pump inhibitor having inhibitory action against ATP-binding cassette sub-family B member 1 (ABCB1), ATP-binding cassette sub-family G member 2 (ABCG2), or having dual inhibitory action against both ABCB1 and ABCG2.

8. The method of claim 2 , further comprising isolating the identified cells exhibiting increased fluorescence.

9. The method of claim 2 , wherein the mixed population of include stem cells.

Assignees

• Univ Johns Hopkins

Inventors

• Pomper Martin G
• Wang Haofan
• Minn Il
• Leach Steven D
• Mease Ronnie C

Classifications

• C07F5/022

  without C-boron linkages · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• C07D207/44 Primary

  having three double bonds between ring members or between ring members and non-ring members · CPC title