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RatioWorks™ PH165, NHS ester

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.
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Physical properties
Molecular weight581.51
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 NHS ester is a novel fluorescent probe that can be used to measure intracellular pH in live cells. It shows pH-dependent fluorescence spectra. Its dual excitation and dual emission make it a unique ratiometric pH probe for analyzing live cells. At low pH, RatioWorks™ PH165 is weakly fluorescent in far red spectrum region and highly fluorescent in red spectrum region. As pH increases, it shows higher fluorescence in far red spectrum and lower fluorescence in red spectrum. This probe can quantify cytosolic pH in the range from 4 to 9 with dual excitation/emission at 497/594 nm and 578/654 nm respectively. The pKa of PH165 is ~7.1. The red fluorescence spectrum of RatioWorks™ PH165 makes it suitable for multiplexing tests with green fluorophores such as GFP. The amine-reactive RatioWorks™ PH165 NHS ester is an excellent building block for developing novel ratiometric fluorescent probes of a variety of biological applications that require the precise pH determinations with the minimal effect of pH probe concentration change.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of RatioWorks™ PH165, NHS ester 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 mM171.966 µL859.83 µL1.72 mL8.598 mL17.197 mL
5 mM34.393 µL171.966 µL343.932 µL1.72 mL3.439 mL
10 mM17.197 µL85.983 µL171.966 µL859.83 µL1.72 mL

Molarity calculator

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Spectral properties

Excitation (nm)463
Emission (nm)641



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