Methods of using fluorescent pH indicators for intracellular assays
View Patent ↗Systems, including compositions and methods, for measuring pH, particularly in cells, organelles, and other samples. The compositions include pH-sensitive fluorescent and fluorogenic 2′,7′-dialkylfluorescein derivatives and associated nonfluorescent precursor compounds. The compositions may permit ratiometric measurement in the excitation spectrum and the emission spectrum. The methods include adding a precursor compound to a sample cell, incubating the sample cell to release the free indicator, illuminating the sample cell, and detecting the fluorescence response of the free indicator.
1. A method of staining a biological sample, comprising the steps of:
a) adding a first compound to the sample, where the first compound having the formula
wherein R 1 and R 2 are independently C 1 -C 12 alkyl that is optionally substituted one or more times by C 1 -C 6 alkoxy or halogen, provided that the alpha-carbon of the alkyl is not halogenated;
R 3 and R 4 are independently C 1 -C 6 alkyl;
R 5 -R 8 are independently hydrogen, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, sulfonic acid, ester of sulfonic acid, carboxylic acid, ester of carboxylic acid, -L-R X , or -L-S C , where L is a covalent linkage, R X is a reactive functional group, and S C is a conjugated substance;
provided that at least one of R 5 -R 8 is sulfonic acid, ester of sulfonic acid, carboxylic acid, ester of carboxylic acid, -L-R X , or -L-S C ; and
b) incubating the sample for a time sufficient for at least some of the first compound to be converted to a second compound having the formula
wherein R 1 -R 8 are as defined above, thereby staining the sample.
2. The method of claim 1 , further comprising the steps of:
c) illuminating the sample at a wavelength suitable to excite the second compound; and
d) detecting a fluorescence response from the second compound.
3. The method of claim 2 , further comprising correlating the fluorescence response with the pH of the sample.
4. The method of claim 1 , wherein the sample comprises cells.
5. The method of claim 4 , wherein the step of adding the first compound comprises incubating the sample cells with the first compound.
6. The method of claim 1 , wherein the first compound is converted to the second compound by esterase enzymes.
7. The method of claim 1 , wherein the fluorescence response is detected using a microplate reader, a flow cytometer, or a fluorescence microscope.
8. The method of claim 1 , further comprising adding an additional detection reagent to the sample.
9. The method of claim 1 , further comprising adding to the sample a substantially cell-impermeant dye capable of accepting fluorescence energy transfer from the second compound.