AAT Bioquest

RatioWorks™ PH165

The pH dependent absorbance spectra of  RatioWorks™ PH165.
The pH dependent absorbance spectra of  RatioWorks™ PH165.
The pH dependent absorbance spectra of  RatioWorks™ PH165.
The pH dependent Emission spectra of RatioWorks™ PH165.
The pH dependent Excitation spectra of  RatioWorks™ PH165.
Ordering information
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Physical properties
Molecular weight517.54
Spectral properties
Excitation (nm)463
Emission (nm)641
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
RatioWorks™ PH165 is a novel fluorescent probe designed for pH measurement in solution, featuring pH-dependent fluorescence spectra and unique dual excitation and dual emission properties. At low pH, RatioWorks™ PH165 is weakly fluorescent in the far-red spectrum region and highly fluorescent in the red spectrum region. As pH increases, it shows higher fluorescence in the far-red spectrum and lower fluorescence in the red spectrum. This probe quantifies pH in the 4 to 9 range, utilizing dual excitation/emission at 497/594 nm and 578/654 nm, respectively, and has a pKa of ~7.1. The red fluorescence spectrum of RatioWorks™ PH165 makes it suitable for multiplexing tests with green fluorophores like GFP. RatioWorks™ PH165 AM is cell-permeable and compatible with platforms such as fluorescence microscopy and microplate readers.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of RatioWorks™ PH165 to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM193.222 µL966.109 µL1.932 mL9.661 mL19.322 mL
5 mM38.644 µL193.222 µL386.444 µL1.932 mL3.864 mL
10 mM19.322 µL96.611 µL193.222 µL966.109 µL1.932 mL

Molarity calculator

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Spectral properties

Excitation (nm)463
Emission (nm)641

Product Family

NameExcitation (nm)Emission (nm)
RatioWorks™ PDMPO333531



View all 3 citations: Citation Explorer
Intracellular pH Measurements in Glioblastoma Cells Using the pH-Sensitive Dye BCECF
Authors: Galifianakis, N. V., Placantonakis, D. G., Chesler, M.
Journal: Methods Mol Biol (2018): 103-109
Evaluation of BCECF fluorescence ratio imaging to properly measure gastric intramucosal pH variations in vivo
Authors: Rochon, P., Jourdain, M., Mangalaboyi, J., Fourrier, F., Soulie-Begu, S., Buys, B., Dehlin, G., Lesage, J. C., Chambrin, M. C., Mordon, S.
Journal: J Biomed Opt (2007): 064014
Calibration methods and avoidance of errors in measurement of intracellular pH (pHcyt) using the indicator bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in human platelets
Authors: Valant, P. A., Haynes, D. H.
Journal: J Fluoresc (1992): 191-206


View all 1 references: Citation Explorer
Estimation of intramitochondrial pca and ph by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (bcecf) fluorescence
Authors: Davis MH, Altschuld RA, Jung DW, Brierley GP.
Journal: Biochem Biophys Res Commun (1987): 40