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AAT Bioquest

Indo-1, AM *UltraPure Grade* *CAS 112926-02-0*

Fluorescence emission spectra of Indo-1 in solutions conctaining 0 to 39uM free Ca2+.
Fluorescence emission spectra of Indo-1 in solutions conctaining 0 to 39uM free Ca2+.
Fluorescence emission spectra of Indo-1 in solutions conctaining 0 to 39uM free Ca2+.
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Physical properties
Dissociation constant (Kd, nM)230
Molecular weight1009.91
SolventDMSO
Spectral properties
Extinction coefficient (cm -1 M -1)33000
Excitation (nm)330
Emission (nm)404
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Alternative formats
Indo-1, AM *CAS 112926-02-0*

OverviewpdfSDSpdfProtocol


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
Calcium measurements are critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding to Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. This cell-permeant Indo-1 AM *UltraPure Grade*, is a UV light excitable, emission ratioable Ca2+ indicator. Upon binding to Ca2+, the emission maximum of Indo-1 AM shifts from 480 nm to 400 nm. Indo-1 is preferred for flow cytometry, in which it is more practical to use a single laser for excitation, such as the 351-364 nm spectral lines of the argon-ion laser.

Platform


Fluorescence microscope

ExcitationIndo-1 filter set
EmissionIndo-1 filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation340
Emission400, 475
CutoffEx/Em = 340/400, no cut off. Ex/Em = 340/475, cut off 455
Recommended plateBlack 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.

Indo-1 AM *UltraPure Grade* Stock Solution
Prepare a 2 to 5 mM stock solution of Indo-1 AM in high-quality, anhydrous DMSO.   

PREPARATION OF WORKING SOLUTION

Indo-1 AM *UltraPure Grade* 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 dye working solution of 2 to 20 µM 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 solution, 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.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Indo-1 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.
  3. 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.
  4. 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.
  5. 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

Table 1. Volume of DMSO needed to reconstitute specific mass of Indo-1, AM *UltraPure Grade* *CAS 112926-02-0* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM99.019 µL495.094 µL990.187 µL4.951 mL9.902 mL
5 mM19.804 µL99.019 µL198.037 µL990.187 µL1.98 mL
10 mM9.902 µL49.509 µL99.019 µL495.094 µL990.187 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)33000
Excitation (nm)330
Emission (nm)404

Images


Citations


View all 5 citations: Citation Explorer
Small-molecule--based generation of functional cardiomyocytes from human umbilical cord--derived induced pluripotent stem cells
Authors: Wu, Kai Hong and Wang, Su Yun and Xiao, Qian Ru and Yang, Yu and Huang, Ning Ping and Mo, Xu Ming and Sun, Jian
Journal: Journal of cellular biochemistry (2018)
Nifedipine stimulates proliferation and migration of different breast cancer cells by distinct pathways
Authors: Zhao, Tao and Guo, Dongqing and Gu, Yuchun and Ling, Yang
Journal: Molecular Medicine Reports (2017): 2259--2263
Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension
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
Nifedipine promotes the proliferation and migration of breast cancer cells
Authors: Guo, Dong-Qing and Zhang, Hao and Tan, Sheng-Jiang and Gu, Yu-Chun
Journal: PloS one (2014): e113649

References


View all 84 references: Citation Explorer
Measurement of [Ca2+] in cell suspensions using indo-1
Authors: Nelemans A., undefined
Journal: Methods Mol Biol (2006): 47
A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence
Authors: Eerbeek O, Mik EG, Zuurbier CJ, van 't Loo M, Donkersloot C, Ince C.
Journal: J Appl Physiol (2004): 2042
Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes
Authors: Sakurai K, Norota I, Tanaka H, Kubota I, Tomoike H, Endo M.
Journal: Life Sci (2002): 1173
Usefulness of the analytic method of intracellular calcium and the problems--aequorin and indo-1 signal
Authors: Endoh M., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 361
Comment on "Usefulness of intracellular calcium analysis and the problem--aequorin and indo-1 signal"
Authors: Imaizumi Y., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 101
Concentrations of caffeine greater than 20 mM increase the indo-1 fluorescence ratio in a Ca(2+)-independent manner
Authors: McKemy DD, Welch W, Airey JA, Sutko JL.
Journal: Cell Calcium (2000): 117
Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice
Authors: Collet C, Allard B, Tourneur Y, Jacquemond V.
Journal: J Physiol (1999): 417
Measurement of [Ca2+]i in cell suspensions using indo-1
Authors: Nelemans A., undefined
Journal: Methods Mol Biol (1999): 41
Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures
Authors: Wang SQ, Zhou ZQ.
Journal: Life Sci (1999): 871