logo
AAT Bioquest

Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*

Product Image
Product Image
Gallery Image 1
Ordering information
Price
Catalog Number
AvailabilityIn stock
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Physical properties
Dissociation constant (Kd, nM)345
Molecular weight1096.95
SolventDMSO
Spectral properties
Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161
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
Related products
Chemical Phosphorylation Reagent I (CPR I)
Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
Cal Green™ 1, hexapotassium salt
Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]
Cal-590™-Dextran Conjugate *MW 3,000*
Cal-590™-Dextran Conjugate *MW 10,000*
Cal-590™ AM
Cal-590™, sodium salt
Cal-590™, potassium salt
Cal-630™ AM
Cal-630™, sodium salt
Cal-630™, potassium salt
Cal-630™-Dextran Conjugate *MW 3,000*
Cal-630™-Dextran Conjugate *MW 10,000*
Fluo-4, Pentapotassium Salt
Cal Red™ R525/650 potassium salt
Cal Red™ R525/650 AM
Cal-520®-Dextran Conjugate *MW 3,000*
Cal-520®-Dextran Conjugate *MW 10,000*
Cal-520®-Biotin Conjugate
Cal-520®-Biocytin Conjugate
Cal-520® NHS Ester
Cal-520® maleimide
Fluo-3, pentasodium salt
Fluo-3, pentapotassium salt
Fluo-3, pentaammonium salt
Fluo-3FF, pentapotassium salt
Fluo-8®, sodium salt
Fluo-8®, potassium salt
Fluo-8H™, sodium salt
Fluo-8L™, sodium salt
Fluo-8L™, potassium salt
Fluo-8FF™, potassium salt
Cal-520®, sodium salt
Cal-520®, potassium salt
Cal-520FF™, AM
Cal-520FF™, potassium salt
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit
Mag-Fluo-4 potassium salt
Mag-Fluo-4 AM
Fluo-2, potassium salt
Fluo-5F, pentapotassium Salt *Cell impermeant*
Fluo-5N, pentapotassium Salt *Cell impermeant*
Cal-520N™, AM
Cal-520N™, potassium salt
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit
Calbryte™ 520, potassium salt
Calbryte™ 590 AM
Calbryte™ 590, potassium salt
Calbryte™ 630 AM
Calbryte™ 630, potassium salt
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit
Calbryte™-520L AM
Calbryte™-520L, potassium salt
Cal-500™, potassium salt
Cal-500™ AM
Cal-670™, potassium salt
Cal-670™-Dextran Conjugate *MW 3,000*
Cal-670™-Dextran Conjugate *MW 10,000*
Cal-770™, potassium salt
Cal-770™-Dextran Conjugate *MW 3,000*
Cal-770™-Dextran Conjugate *MW 10,000*
Calbryte™-520XL azide
Calbryte™-520XL, potassium salt
Calbryte™-520XL AM
Calbryte™-520XL-Dextran
Cal-520L®-Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-520L®/Cy5-Dextran Conjugate *MW 10,000*
Cal-520L™ maleimide
RatioWorks™ Cal-520®/zFluor 647™ -Dextran Conjugate *MW 10,000*
Cal-590L® Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-590L®/Cy5-Dextran Conjugate *MW 10,000*
Cal-520® amine
Cal-520® azide
Cal-520® alkyne
Show More (67)

OverviewpdfSDSpdfProtocol


CAS
273221-67-3
Molecular weight
1096.95
Dissociation constant (Kd, nM)
345
Extinction coefficient (cm -1 M -1)
82000
Excitation (nm)
495
Emission (nm)
528
Quantum yield
0.161
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Fluo-4 are most commonly used among the visible light-excitable calcium indicators. Fluo-4 is an analog of Fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis, and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8® and Cal-520® calcium dyes have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation @ ~490 nm and maximum emission @ ~520 nm.

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

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

Fluo-4 AM *UltraPure grade* Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fluo-4 AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fluo-4 AM *UltraPure grade* Stock Solution
  1. On the day of the experiment, either dissolve Fluo-4 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Fluo-4 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, Fluo-4 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 Fluo-4 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 Fluo-4 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 2 hours 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 490/525 nm cutoff 515 nm.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* 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 mM91.162 µL455.809 µL911.619 µL4.558 mL9.116 mL
5 mM18.232 µL91.162 µL182.324 µL911.619 µL1.823 mL
10 mM9.116 µL45.581 µL91.162 µL455.809 µL911.619 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-3, AM *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *Bulk package* *CAS 121714-22-5*50651586,00010.151
Fluo-3FF, AM *UltraPure grade* *Cell permeant*50651586,00010.151
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--
Rhod-4™, AM523551-0.11
Show More (1)

Images


Citations


View all 26 citations: Citation Explorer
Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity
Authors: Shen, Qiaochu and Hasegawa, Keiichi and Oelerich, Nicole and Prakken, Anna and Tersch, Lea Weiler and Wang, Junli and Reichhardt, Frowin and Tersch, Alexandra and Choo, Je Cuan and Timmers, Ton and others,
Journal: Cell Host \& Microbe (2024)
Contrary effects of the gut metabolites deoxycholate and butyrate on the acetylcholine-evoked calcium response in an enteroendocrine cell model
Authors: Pfanzagl, Beatrix and Jensen-Jarolim, Erika
Journal: Endocrine and Metabolic Science (2024): 100167
Biological and mutational analyses of CXCR4--antagonist interactions and design of new antagonistic analogs
Authors: Meng, Qian and Zhu, Ruohan and Mao, Yujia and Zhu, Siyu and Wu, Yi and Huang, Lina SM and Ciechanover, Aaron and An, Jing and Xu, Yan and Huang, Ziwei
Journal: Bioscience Reports (2023)
P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2
Authors: Goretzko, Jonas and Pauels, Inga and Heitzig, Nicole and Thomas, Katharina and Kardell, Marina and Na{\ss}, Johannes and Krogsaeter, Einar Kleinhans and Schloer, Sebastian and Spix, Barbara and Matos, Anna L{\'\i}via Linard and others,
Journal: Cell Reports (2023): 113501
Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1
Authors: Shiba, Naoko and Yang, Xiao and Sato, Mitsuto and Kadota, Shin and Suzuki, Yota and Agata, Masahiro and Nagamine, Kohei and Izumi, Masaki and Honda, Yusuke and Koganehira, Tomoya and others,
Journal: Molecular Therapy-Nucleic Acids (2023)
Microglial Piezo1 senses Ab fibril stiffness to restrict Alzheimer’s disease
Authors: Hu, Jin and Chen, Qiang and Zhu, Hongrui and Hou, Lichao and Liu, Wei and Yang, Qihua and Shen, Huidan and Chai, Guolin and Zhang, Boxin and Chen, Shaoxuan and others,
Journal: Neuron (2023): 1--15
Effects of melatonin on dopaminergic neuron development via IP3-mediated mitochondrial Ca2+ regulation in autism spectrum disorder
Authors: Dong, Shuangshan and Kifune, Takashi and Kato, Hiroki and Wang, Lu and Kong, Jun and Hirofuji, Yuta and Sun, Xiao and Sato, Hiroshi and Ito, Yosuke and Kato, Takahiro A and others,
Journal: Biochemical and Biophysical Research Communications (2023)
HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop
Authors: Zhu, Ruohan and Meng, Qian and Zhang, Huijun and Zhang, Ge and Huang, Lina SM and Xu, Yan and Schooley, Robert T and An, Jing and Huang, Ziwei
Journal: European Journal of Medicinal Chemistry (2022): 114797
Tetralol derivative NNC-55-0396 induces glioblastoma cell death by activating IRE1$\alpha$, JNK1 and calcium signaling
Authors: Visa, Anna and Alza, L{\'\i}a and Cant{\'\i}, Carles and Herreros, Judit
Journal: Biomedicine \& Pharmacotherapy (2022): 112881
Functional Analysis of Liposomes Containing Single-Walled Carbon Nanotubes (SWNTs) by Fluorescence Microscopy
Authors: Kanno, Shoichiro and Peng, Zugui and Shimba, Kenta and Miyamoto, Yoshitaka and Yagi, Tohru
Journal: IEEJ Transactions on Electronics, Information and Systems (2021): 620--626