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Fluo-8L™, AM

U2OS cells were seeded overnight at 40,000 cells per 100 uL per well in a 96-well black all/clear bottom costar plate.  The growth medium was removed, and the cells were incubated with 100 uL of 4 uM Fluo-3 AM, Fluo-4 AM or Fluo-8® AM in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 uL HHBS, then imaged with a fluorescence microscope using FITC channel.
U2OS cells were seeded overnight at 40,000 cells per 100 uL per well in a 96-well black all/clear bottom costar plate.  The growth medium was removed, and the cells were incubated with 100 uL of 4 uM Fluo-3 AM, Fluo-4 AM or Fluo-8® AM in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 uL HHBS, then imaged with a fluorescence microscope using FITC channel.
Ordering information
Price ()
Catalog Number21096
Unit Size
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Additional ordering information
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ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)1900
Molecular weight1078.95
Spectral properties
Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
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
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Molecular weight
Dissociation constant (Kd, nM)
Correction Factor (260 nm)
Correction Factor (280 nm)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
Calcium measurements are critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy, and fluorescence microplate readers. Fluo-3 AM and Fluo-4 AM are most commonly used among the visible light-excitable calcium indicators for live-cell calcium imaging. 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® dyes are developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of Ex/Em = ∼490/∼520 nm. Fluo-8® AM can be loaded into cells at room temperature, while Fluo-3 AM and Fluo-4 AM require 37°C for cell loading. In addition, Fluo-8® AM is two times brighter than Fluo-4 AM and four times brighter than Fluo-3 AM. AAT Bioquest offers a set of our outstanding Fluo-8® reagents with different calcium-binding affinities (Fluo-8® Kd = 389 nM; Fluo-8H™ Kd = 232 nM; Fluo-8L™ Kd = 1.86 µM; Fluo-8FF™ Kd = 10 µM). We also offer versatile packing sizes to meet your special needs (e.g., 1 mg, 10x50 µg, 20x50 µg, and HTS packages) with no additional packaging charge.


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.

Fluo-8L™ AM Stock Solution
Prepare a 2 to 5 mM stock solution of Fluo-8L™ AM in high-quality, anhydrous DMSO.


Fluo-8L™ AM Working Solution
On the day of the experiment, either dissolve Fluo-8L™ 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 buLer of your choice (e.g., Hanks and Hepes buLer) with 0.04% Pluronic® F-127. For most cell lines, Fluo-8L™ 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-8L™ 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.


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-8L™ 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 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. 


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-8L™, 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 mM92.683 µL463.414 µL926.827 µL4.634 mL9.268 mL
5 mM18.537 µL92.683 µL185.365 µL926.827 µL1.854 mL
10 mM9.268 µL46.341 µL92.683 µL463.414 µL926.827 µL

Molarity calculator

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

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Spectral properties

Correction Factor (260 nm)1.076
Correction Factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)23430
Excitation (nm)495
Emission (nm)516
Quantum yield0.161


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