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AAT Bioquest

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

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.

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

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.

PREPARATION OF WORKING SOLUTION

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.

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 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.

Calculators

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
/=x=

Spectrum

Citations

View all 5 citations: Citation Explorer
Protocol for in vitro sonoporation validation using non-targeted microbubbles for human studies of ultrasound-mediated gene delivery
Authors: Zhang, Nisi and Guo, Yutong and Foiret, Josquin and Tumbale, Spencer K and Paulmurugan, Ramasamy and Ferrara, Katherine W
Journal: STAR protocols (2023): 102723
Network pharmacology-based research uncovers cold resistance and thermogenesis mechanism of Cinnamomum cassia
Authors: Jiang, Xiao-wen and Lu, Hong-yuan and Xu, Zi-Hua and Zhang, Ying-Shi and Zhao, Qing-Chun and others,
Journal: Fitoterapia (2021): 104824
Binding to carboxypeptidase M mediates protective effects of fibrinopeptide Bβ (15-42)
Authors: Sörensen-Zender, Inga and Chen, Rongjun and Song, Rong and David, Sascha and Melk, Anette and Haller, Hermann and Schmitt, Rol and , undefined
Journal: Translational Research (2019)
Tuning the color palette of fluorescent copper sensors through systematic heteroatom substitution at rhodol cores
Authors: Jia, Shang and Ramos-Torres, Karla M and Kolemen, Safacan and Ackerman, Cheri M and Chang, Christopher J
Journal: ACS chemical biology (2017): 1844--1852
Tuning the Color Palette of Fluorescent Copper Sensors through Systematic Heteroatom Substitution at Rhodol Cores
Authors: Jia, Shang and Ramos-Torres, Karla M and Kolemen, Safacan and Ackerman, Cheri M and Chang, Christopher J
Journal: (2017)

References

View all 11 references: Citation Explorer
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
Authors: Silei V, Fabrizi C, Venturini G, Tagliavini F, Salmona M, Bugiani O, Lauro GM.
Journal: Brain Res Brain Res Protoc (2000): 132
Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran
Authors: McPherson DR, McClellan AD, O'Donovan MJ.
Journal: Brain Res Brain Res Protoc (1997): 157
Page updated on November 9, 2024

Ordering information

Price
Unit size
10x50 ug
1 mg
Catalog Number
2050120502
Quantity
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Additional ordering information

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Physical properties

Dissociation constant (Kd, nM)190

Molecular weight

1290.96

Solvent

DMSO

Spectral properties

Excitation (nm)

498

Emission (nm)

517

Quantum yield

0.751

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

CAS

186501-28-0

Platform

Fluorescence microscope

ExcitationFITC
EmissionFITC
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling