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Protonex™ Red 670 AM *Cell-Permeable*

Protonex™ Red 670 AM is the cell-permeable version of Protonex™ Red 670. Protonex™ Red 670 dye works by changing its fluorescence intensity depending on the pH of the environment. Protonex™ Red 670 is minimally fluorescent at a basic pH and maximally fluorescent at an acidic pH. When Protonex™ Red 670 is bound to an acidic intracellular target, it becomes highly fluorescent and emits red light when excited by a red laser such as a 632 nm He-Ne or 647 nm red laser. By measuring the fluorescence intensity of Protonex™ Red 670, one can label or monitor the acidic intracellular targets in live cells.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Protonex™ Red 670 AM Stock Solution
  1. Prepare a 10 to 20 mM stock solution of Protonex™ Red 670 AM in high-quality anhydrous DMSO.

    Note: An unused Protonex™ Red 670 AM stock solution should be divided into single-use aliquots and stored at ≤ -20 º C.  Protect from light and avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Protonex™ Red 670 AM Working Solution
  1. On the day of the experiment, either dissolve Protonex™ Red 670 AM dye in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a Protonex™ Red 670 AM dye working solution of 5 to 20 μM in a buffer of your choice (e.g., Hanks and Hepes buffer).

    Note: The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. The final Pluronic® F-127 concentration should be approximately 0.02% in the staining buffer. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest (here). Avoid long-term storage of AM esters in the presence of Pluronic® F-127.

    Note: If your cells contain organic anion-transporters, probenecid (1-4 mM) may be added to the dye working solution (final in well concentration will be 0.5-2 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest (here). 

SAMPLE EXPERIMENTAL PROTOCOL

Important

The following is a recommended protocol for loading Protonex™ Red 670 AM dye into live mammalian cells. This protocol only provides a guideline and should be modified according to your specific needs.

  1. Prepare viable cells as desired (e.g., 100 µL/well/96-well plate or 25 µL/well/384-well plate).

  2. On the next day, add the Protonex™ Red 670 AM dye working solution into the cell plate in equal volumes, such as 100 µL/well/96-well plate or 25 µL/well/384-well plate.

  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.

  4. Replace the dye working solution with HHBS or buffer of your choice to remove any excess dye.

  5. Prepare the compound plates using HHBS or a buffer of your choice.

  6. Perform the pH assay as desired while simultaneously monitoring fluorescence. This can be done with either a fluorescence microscope that has a Cy5 filter set or a fluorescence plate reader at Ex/Em = 640/680 nm (cutoff 665 nm).

Spectrum

References

View all 50 references: Citation Explorer
Mechanisms of 15-Epi-LXA4-Mediated HO-1 in Cytoprotection Following Inflammatory Injury.
Authors: Wang, Meng and Tong, Kun and Chen, Zhe and Wen, Zhengde
Journal: The Journal of surgical research (2023): 245-255
mOrange2, a Genetically Encoded, pH Sensitive Fluorescent Protein, is an Alternative to BCECF-AM to Measure Intracellular pH to Determine NHE3 and DRA Activity.
Authors: Sarker, Rafiquel and Tse, Chung Ming and Lin, Ruxian and McNamara, George and Singh, Varsha and Donowitz, Mark
Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology (2022): 39-49
The role of GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses counteracted by PPARγ agonists.
Authors: Miao, Yumeng and Zheng, Yun and Geng, Yanzhi and Yang, Ling and Cao, Na and Dai, Yue and Wei, Zhifeng
Journal: Theranostics (2021): 4531-4548
Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm.
Authors: He, Qifu and Wu, Shenghui and Huang, Ming and Wang, Ying and Zhang, Kang and Kang, Jian and Zhang, Yong and Quan, Fusheng
Journal: Frontiers in cell and developmental biology (2021): 747722
Role of growth hormone signaling pathways in the development of atherosclerosis.
Authors: Ishikawa, Mayumi and Toyomura, Junko and Yagi, Takashi and Kuboki, Koji and Morita, Toshisuke and Sugihara, Hitoshi and Hirose, Takahisa and Minami, Shiro and Yoshino, Gen
Journal: Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Socie (2020): 101334
Page updated on December 11, 2024

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Catalog Number21182
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Physical properties

Molecular weight

521.05

Solvent

DMSO

Spectral properties

Excitation (nm)

643

Emission (nm)

660

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationCy5 Filter Set
EmissionCy5 Filter Set
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation640 nm
Emission680 nm
Cutoff665 nm
Recommended plateBlack wall, clear bottom
Response of HeLa cells labeled with Protonex™ Red 670 AM. HeLa cells were incubated with 5 µM of Protonex™ Red 670 AM for 30 minutes at 37°C. Incubation of Protonex™ Red 670 AM solution with HeLa cells showed a homogenous uptake of Protonex™ Red 670 AM and stained cell cytosol. The Spexyte™ Intracellular pH Calibration Buffer Kit (Cat No. 21235) was used to clamp the intracellular pH with extracellular buffers at pH 4 to 10. (A) Images were acquired using a fluorescence microscope with a Cy5 filter set, and (B) fluorescence was measured using a ClarioStar fluorescence microplate reader (Ex/Em = 640/680 nm, cutoff = 665 nm).
Response of HeLa cells labeled with Protonex™ Red 670 AM. HeLa cells were incubated with 5 µM of Protonex™ Red 670 AM for 30 minutes at 37°C. Incubation of Protonex™ Red 670 AM solution with HeLa cells showed a homogenous uptake of Protonex™ Red 670 AM and stained cell cytosol. The Spexyte™ Intracellular pH Calibration Buffer Kit (Cat No. 21235) was used to clamp the intracellular pH with extracellular buffers at pH 4 to 10. (A) Images were acquired using a fluorescence microscope with a Cy5 filter set, and (B) fluorescence was measured using a ClarioStar fluorescence microplate reader (Ex/Em = 640/680 nm, cutoff = 665 nm).
Response of HeLa cells labeled with Protonex™ Red 670 AM. HeLa cells were incubated with 5 µM of Protonex™ Red 670 AM for 30 minutes at 37°C. Incubation of Protonex™ Red 670 AM solution with HeLa cells showed a homogenous uptake of Protonex™ Red 670 AM and stained cell cytosol. The Spexyte™ Intracellular pH Calibration Buffer Kit (Cat No. 21235) was used to clamp the intracellular pH with extracellular buffers at pH 4 to 10. (A) Images were acquired using a fluorescence microscope with a Cy5 filter set, and (B) fluorescence was measured using a ClarioStar fluorescence microplate reader (Ex/Em = 640/680 nm, cutoff = 665 nm).