Cal Red™ R525/650 AM
Price | |
Catalog Number | |
Unit Size | |
Quantity |
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 |
Dissociation constant (Kd, nM) | 330 |
Molecular weight | ~1100 |
Solvent | DMSO |
Certificate of Origin | Download PDF |
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 | 12352200 |
Overview | SDSProtocol |
Molecular weight ~1100 | Dissociation constant (Kd, nM) 330 |
Platform
Fluorescence microscope
Excitation | FITC |
Emission | FITC |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 490 |
Emission | 525, 660 |
Cutoff | 515, 630 |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
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 2 to 5 mM stock solution of Cal Red™ R525/650 AM in anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Cal Red™ R525/650 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Cal Red™ R525/650 AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Cal Red™ R525/650 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Cal Red™ R525/650 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 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.
SAMPLE EXPERIMENTAL PROTOCOL
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
- Prepare cells in growth medium overnight.
On the next day, add 1X Cal Red™ R525/650 AM working solution into your cell plate.
Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note: Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
- Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
- Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 490/525 nm cutoff 515 nm and Ex/Em2 = 490/660 nm cutoff 630 nm.
Images
Citations
Authors: Sun, Zhengwu and Lu, Kun and Kamla, Christine and Kameritsch, Petra and Seidel, Thomas and Dendorfer, Andreas
Journal: Communications Biology (2024): 220
Authors: Wang, Xiaobo and Geng, Shuang and Meng, Junchen and Kang, Ning and Liu, Xinyi and Xu, Yanni and Lyu, Huiyun and Xu, Ying and Xu, Xun and Song, Xinrong and others,
Journal: The Journal of Clinical Investigation (2023)
Authors: Wu, Yue and Zhang, Yunshan and Xu, Zhongyuan and Guo, Xinyu and Yang, Wenjian and Zhang, Xiaoyu and Liao, Yuheng and Fan, Minzhi and Zhang, Diming
Journal: Biosensors (2022): 917
Authors: Debattisti, Valentina and Paillard, Melanie and Csordas, Gyorgy and Seifert, Erin and Hajnoczky, Gyorgy
Journal: Biophysical Journal (2016): 259a
Authors: Vadrevu, Suryakiran and Satin, Leslie S
Journal: Biophysical Journal (2016): 258a--259a
Authors: Zorin, Nikolay and Ryvkin, Alexander and Moskvin, Alexander and Solovyova, Olga
Journal: Biophysical Journal (2016): 259a
Authors: Diwu, Zhenjun and Zhao, Qin and Luo, Zhen and Meng, Qinglin and Liu, Jixiang and Liao, Jinfang
Journal: Biophysical Journal (2016): 259a
Authors: Qudrat, Anam and Truong, Kevin
Journal: Biophysical Journal (2016): 259a
Authors: Sirenko, Syevda and Yang, Dongmei and Lakatta, Edward G
Journal: Biophysical Journal (2016): 259a--260a
References
Authors: Wendt ER, Ferry H, Greaves DR, Keshav S.
Journal: PLoS One (2015): e0119532
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Authors: Walczysko P, Wagner E, Albrechtova JT.
Journal: Cell Calcium (2000): 23
Authors: Su ZL, Li N, Sun YR, Yang J, Wang IM, Jiang SC.
Journal: Shi Yan Sheng Wu Xue Bao (1998): 323
Authors: Blackwood AM, Sagnella GA, Mark and u ND, MacGregor GA.
Journal: J Hum Hypertens (1997): 601
Authors: Wu Y, Clusin WT.
Journal: Am J Physiol (1997): H2161
Authors: Floto RA, Mahaut-Smith MP, Somasundaram B, Allen JM.
Journal: Cell Calcium (1995): 377
Authors: Schild D, Jung A, Schultens HA.
Journal: Cell Calcium (1994): 341
Authors: Novak EJ, Rabinovitch PS.
Journal: Cytometry (1994): 135
Authors: Lew VL, Etzion Z, Bookchin RM, daCosta R, Vaananen H, Sassaroli M, Eisinger J.
Journal: Biochim Biophys Acta (1993): 152
Application notes
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Evaluation of FLIPR Calcium Assays for Screening GPCR and Calcium Channel Targets
Fluorescent Dye AM Esters
FAQ
How do I make an AM ester stock solution?
How do you measure Ca2+ response with calcium indicators like Calbryte 520, potassium salt?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?