Protonex™ Lyso-Red 670
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
Prepare a 10 to 20 mM stock solution of Protonex™ Lyso-Red 670 in high-quality anhydrous DMSO.
Note: An unused Protonex™ Lyso-Red 670 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
On the day of the experiment, either dissolve Protonex™ Lyso-Red 670 in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a Protonex™ Lyso-Red 670 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. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. 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
The following is a recommended protocol for loading Protonex™ Lyso-Red 670 into live mammalian cells. This protocol only provides a guideline and should be modified according to your specific needs.
Prepare viable cells as desired {eg. 100 µL/well (96-well plate) or 25 µL/well (384-well plate)}.
On the next day, add equal volumes, such as 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of the Protonex™ Lyso-Red 670 working solution, into the cell plate.
Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Replace the dye working solution with HHBS or buffer of your choice to remove any excess probes.
Prepare the compound plates using HHBS or a buffer of your choice.
Run the pH assay as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a Cy5 filter set or a fluorescence plate reader at Ex/Em = 640/680 nm cutoff 665 nm.
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) |
Protonex™ Red 670 acid | 643 | 660 |
Protonex™ Red 670 AM *Cell-Permeable* | 643 | 660 |
References
Authors: Mattison, Kari A and Tossing, Gilles and Mulroe, Fred and Simmons, Callum and Butler, Kameryn M and Schreiber, Alison and Alsadah, Adnan and Neilson, Derek E and Naess, Karin and Wedell, Anna and Wredenberg, Anna and Sorlin, Arthur and McCann, Emma and Burghel, George J and Menendez, Beatriz and Hoganson, George E and Botto, Lorenzo D and Filloux, Francis M and Aledo-Serrano, Ángel and Gil-Nagel, Antonio and Tatton-Brown, Katrina and Verbeek, Nienke E and van der Zwaag, Bert and Aleck, Kyrieckos A and Fazenbaker, Andrew C and Balciuniene, Jorune and Dubbs, Holly A and Marsh, Eric D and Garber, Kathryn and Ek, Jakob and Duno, Morten and Hoei-Hansen, Christina E and Deardorff, Matthew A and Raca, Gordana and Quindipan, Catherine and van Hirtum-Das, Michele and Breckpot, Jeroen and Hammer, Trine Bjørg and Møller, Rikke S and Whitney, Andrea and Douglas, Andrew G L and Kharbanda, Mira and Brunetti-Pierri, Nicola and Morleo, Manuela and Nigro, Vincenzo and May, Halie J and Tao, James X and Argilli, Emanuela and Sherr, Elliot H and Dobyns, William B and , and Baines, Richard A and Warwicker, Jim and Parker, J Alex and Banka, Siddharth and Campeau, Philippe M and Escayg, Andrew
Journal: Brain : a journal of neurology (2023): 1357-1372
Authors: Chen, Deshu and Yu, Wenjie and Zhong, Chongbin and Hong, Qingqing and Huang, Guanlin and Que, Dongdong and Wang, Yuxi and Yang, Yashu and Rui, Bowen and Zhuang, Zhenyu and Liang, Miaoyuan and Ye, Zhicheng and Yan, Xin and Lv, Jiankun and Zhang, Ronghua and Yan, Jing and Yang, Pingzhen
Journal: Pharmacological research (2022): 106186
Authors: Hong, Seong-Hwi and Lee, Kwang-Suk and Hwang, Hyun-Ji and Park, Sung-Yul and Han, Woong-Kyu and Yoon, Young-Eun
Journal: Genes (2022)
Authors: Sage, Stewart O and Harper, Alan G S
Journal: Cell calcium (2022): 102522
Authors: Allan, Claire Y and Fisher, Paul R
Journal: Frontiers in cell and developmental biology (2022): 741967