Indo-1, AM *CAS 112926-02-0*
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Dissociation constant (Kd, nM) | 230 |
Molecular weight | 1009.91 |
Solvent | DMSO |
Extinction coefficient (cm -1 M -1) | 33000 |
Excitation (nm) | 330 |
Emission (nm) | 404 |
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 |
Indo-1, AM *UltraPure Grade* *CAS 112926-02-0* |
Overview | SDSProtocol |
CAS 112926-02-0 | Molecular weight 1009.91 | Dissociation constant (Kd, nM) 230 | Extinction coefficient (cm -1 M -1) 33000 | Excitation (nm) 330 | Emission (nm) 404 |
Platform
Fluorescence microscope
Excitation | Indo-1 filter set |
Emission | Indo-1 filter set |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 340 |
Emission | 400, 475 |
Cutoff | Ex/Em = 340/400, no cut off. Ex/Em = 340/475, cut off 455 |
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 Indo-1 AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Indo-1 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Indo-1 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, Indo-1 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 Indo-1 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 Indo-1 AM working solution to 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 an Indo-1 filter set or a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 340/400 nm no cutoff and Ex/Em2 = 340/475 cutoff 455 nm.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 99.019 µL | 495.094 µL | 990.187 µL | 4.951 mL | 9.902 mL |
5 mM | 19.804 µL | 99.019 µL | 198.037 µL | 990.187 µL | 1.98 mL |
10 mM | 9.902 µL | 49.509 µL | 99.019 µL | 495.094 µL | 990.187 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Spectral properties
Extinction coefficient (cm -1 M -1) | 33000 |
Excitation (nm) | 330 |
Emission (nm) | 404 |
Images
Citations
Authors: Birkner, Antje
Journal: (2019)
Authors: Zhao, Tao and Guo, Dongqing and Gu, Yuchun and Ling, Yang
Journal: Molecular Medicine Reports (2017): 2259--2263
Authors: Guo, Dongqing and Gu, Junzhong and Jiang, Hui and Ahmed, Asif and Zhang, Zhiren and Gu, Yuchun
Journal: Journal of molecular and cellular cardiology (2016): 179--187
Authors: Guo, Dong-Qing and Zhang, Hao and Tan, Sheng-Jiang and Gu, Yu-Chun
Journal: PloS one (2014): e113649
References
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Journal: J Appl Physiol (2004): 2042
Authors: Sakurai K, Norota I, Tanaka H, Kubota I, Tomoike H, Endo M.
Journal: Life Sci (2002): 1173
Authors: Endoh M., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 361
Authors: Imaizumi Y., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 101
Authors: McKemy DD, Welch W, Airey JA, Sutko JL.
Journal: Cell Calcium (2000): 117
Authors: Collet C, Allard B, Tourneur Y, Jacquemond V.
Journal: J Physiol (1999): 417
Authors: Nelemans A., undefined
Journal: Methods Mol Biol (1999): 41
Authors: Wang SQ, Zhou ZQ.
Journal: Life Sci (1999): 871
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