AAT Bioquest

Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]

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Physical properties
Dissociation constant (Kd, nM)190
Molecular weight1290.96
Spectral properties
Excitation (nm)498
Emission (nm)517
Quantum yield0.751
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
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Molecular weight
Dissociation constant (Kd, nM)
Excitation (nm)
Emission (nm)
Quantum yield
Cal Green™ 1 (AAT Bioquest) is the same molecule to Calcium Green-1 (Invitrogen). It exhibits an increase in fluorescence intensity upon binding calcium ion. The cell-permeant dye, Cal Green 1 AM (Calcium Green-1 AM) is a 488 nm-excitable calcium indicator. Compared to Fluo-3 AM, Cal Green-1 AM is more fluorescent at low calcium concentrations in cells, facilitating the determination of baseline calcium levels and increasing the visibility of resting cells. It has been used in many calcium signaling investigations, including measuring intracellular calcium, following calcium influx and release, and multiphoton excitation imaging of calcium in living tissues. Cells can be loaded with Cal Green-1 AM by adding the dissolved indicator directly to cultured cells in medium. The fluorescence signal from these cells is generally measured using fluorescence microscopy, fluorescence microplate assays, or flow cytometry.


Fluorescence microscope

Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


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

Cal Green™ 1 AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Cal Green™ 1 AM in high-quality, anhydrous DMSO.


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

  2. Prepare a 2 to 20 µM Cal Green™ 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, Cal Green™ 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 Cal Green™ 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.


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 Cal Green™ 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.

  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 a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM] 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 mM77.462 µL387.309 µL774.617 µL3.873 mL7.746 mL
5 mM15.492 µL77.462 µL154.923 µL774.617 µL1.549 mL
10 mM7.746 µL38.731 µL77.462 µL387.309 µL774.617 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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Spectral properties

Excitation (nm)498
Emission (nm)517
Quantum yield0.751



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Microscopic imaging of intracellular calcium in live cells using lifetime-based ratiometric measurements of Oregon Green BAPTA-1
Authors: Lattarulo C, Thyssen D, Kuchibholta KV, Hyman BT, Bacskaiq BJ.
Journal: Methods Mol Biol (2011): 377
An ensemble and single-molecule fluorescence spectroscopy investigation of Calcium Green 1, a calcium-ion sensor
Authors: Lu Y, Paige MF.
Journal: J Fluoresc (2007): 739
Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator
Authors: Tour O, Adams SR, Kerr RA, Meijer RM, Sejnowski TJ, Tsien RW, Tsien RY.
Journal: Nat Chem Biol (2007): 423
Measurement of intracellular calcium levels by the fluorescent Ca2+ indicator Calcium-Green
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Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran
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Journal: Brain Res Brain Res Protoc (1997): 157
Monitoring of Ca2+ release from intracellular stores in permeabilized rat parotid acinar cells using the fluorescent indicators Mag-fura-2 and calcium green C18
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Journal: Biochem Biophys Res Commun (1997): 189
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Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18
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A method for recording intracellular [Ca2+] transients in cardiac myocytes using calcium green-2
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Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons
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