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RatioWorks™ PH165, AM
Ordering information
| Price | |
| Catalog Number | |
| Unit Size | |
| Quantity |
Additional ordering information
| Telephone | 1-800-990-8053 |
| Fax | 1-800-609-2943 |
| sales@aatbio.com | |
| International | See distributors |
| Bulk request | Inquire |
| Custom size | Inquire |
| Shipping | Standard overnight for United States, inquire for international |
Physical properties
| Molecular weight | 583.53 |
| Solvent | DMSO |
Spectral properties
| Excitation (nm) | 463 |
| Emission (nm) | 641 |
Storage, safety and handling
| H-phrase | H303, H313, H333 |
| Hazard symbol | XN |
| Intended use | Research Use Only (RUO) |
| R-phrase | R20, R21, R22 |
| Storage | Freeze (< -15 °C); Minimize light exposure |
| UNSPSC | 12171501 |
Related products
| Overview |  SDSProtocol |
See also: Intracellular pH
Molecular weight 583.53 | Excitation (nm) 463 | Emission (nm) 641 |
RatioWorks™ PH165 AM 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. RatioWorks™ PH165 AM is cell-permeable, and compatible with various platforms such as fluorescence microscopy and microplate reader.
Platform
Fluorescence microscope
| Excitation | Cy3-Cy5 filter set |
| Emission | Cy3-Cy5 filter set |
| Recommended plate | Black wall/clear bottom |
| Instrument specification(s) | Cy3-Cy5 filter set |
Fluorescence microplate reader
| Excitation | 497-578 nm |
| Emission | 594-654 nm |
| Cutoff | 570-630 nm |
| Recommended plate | Black wall/clear bottom |
| Instrument specification(s) | Bottom read mode |
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of RatioWorks™ PH165, AM to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 171.371 µL | 856.854 µL | 1.714 mL | 8.569 mL | 17.137 mL |
| 5 mM | 34.274 µL | 171.371 µL | 342.742 µL | 1.714 mL | 3.427 mL |
| 10 mM | 17.137 µL | 85.685 µL | 171.371 µL | 856.854 µL | 1.714 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Product Family
| Name | Excitation (nm) | Emission (nm) |
| RatioWorks™ BCFL, AM *Superior replacement for BCECF* | 504 | 527 |
Images
Figure 1. Ratiometric analysis of HeLa cells using RatioWorks™ PH165, AM with fluorescence microscopy. HeLa cells were stained with RatioWorks™ PH165, AM for 90 minutes at 37°C and shifted to solutions with different pH. Images were acquired using Cy3/TRITC (green, pseudo color) and Cy5 filter sets (red color).
Figure 2. Ratiometric analysis of HeLa cells using RatioWorks™ PH165, AM with microplate reader. HeLa cells were stained with RatioWorks™ PH165, AM for 30 minutes at 37°C and shifted to solutions with different pH. RFU was measured using Ex/Em= 497/594 nm and 578/654 nm using fluorescence microplate reader and ratio for 594/654 nm was calculated and plotted.
Citations
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
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
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
Authors: Valant, P. A., Haynes, D. H.
Journal: J Fluoresc (1992): 191-206
References
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
Authors: Davis MH, Altschuld RA, Jung DW, Brierley GP.
Journal: Biochem Biophys Res Commun (1987): 40
Application notes
Cell Loading Protocol For Fluorescent pH Indicator, BCECF-AM
Cell Preparation Considerations and Troubleshooting
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Cell Preparation Considerations and Troubleshooting
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
FAQ
How to measure intracellular pH using flow cytometry?
I am performing a phagocytosis assay of macrophages engulfing Protonex Red 600 labeled tumor cells. How do you recommend I label my cells?
What is an AM ester?
What are the disadvantages of Nanodrop light spectrophotometer?
What are the advantages of a Nanodrop light spectrophotometer?
I am performing a phagocytosis assay of macrophages engulfing Protonex Red 600 labeled tumor cells. How do you recommend I label my cells?
What is an AM ester?
What are the disadvantages of Nanodrop light spectrophotometer?
What are the advantages of a Nanodrop light spectrophotometer?
AssayWise
RATIOWORKS pH 165, AM: A Dual Excitation/Emission Fluorescence Probe For Imaging Live Cells
Intracellular pH Measurement with Dual Excitation Fluorescence Sensor BCFL
Investigating Intercellular Channels: Focus on Gap Junctions
Nucleic Acid Detection, Quantification and Imaging
Cal-520® AM, a Development Overview
Intracellular pH Measurement with Dual Excitation Fluorescence Sensor BCFL
Investigating Intercellular Channels: Focus on Gap Junctions
Nucleic Acid Detection, Quantification and Imaging
Cal-520® AM, a Development Overview