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AAT Bioquest

Fluo-8®, AM

U2OS cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 uM in a 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
U2OS cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 uM in a 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
U2OS cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 uM in a 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
Difference in fluorescence intensity of insect flexion leg muscles of the control beetle and the beetle after oral dosing with chemical indicators. (A) Fluo-8; (B) Rhodamine 123; (C) DiBAC4(3); (D) Rhodamine B; and (E) Cell Tracker. The Fluo-8, Rhodamine 123, and DiBAC4(3) dosed beetle leg was observed under 460&ndash;480 nm excitation light and fluorescence emitted was collected within 495&ndash;540 nm. The Rhodamine B and Cell Tracker dosed beetle leg was observed under 535&ndash;555 nm excitation light and fluorescence emitted was collected within 570&ndash;625 nm. Fluorescence intensity was measured at the 2 regions of interest (ROIs) shown in S1A Fig. The images obtained were digitized by ImageJ software, and the averaged intensity is shown in each bar graph. The graphs in the right column show the fluorescence intensities of each beetle leg dosed with different chemical indicators (center) compared with the control (left) beetle leg. The control beetles were fed with the home-made jelly (no chemical indicator added) for 2 days prior to observation. The error bars represent the standard deviation (S.D.) (N = 5 beetles, n = 30 beetle legs for control, DiBAC4(3), Rhodamine B, and Rhodamine 123; N = 9 beetles, n = 30 beetle legs for Fluo-8 and Cell Tracker). Each data set was compared with control leg data set by student&rsquo;s t-test (Fluo-8, p = 1.42&times;10-4; Rhodamine 123, p = 4.65&times;10-5; DiBAC4(3), p = 6.26&times;10-9; Rhodamine B, p = 1.15&times;10-9 and Cell Tracker, p = 7.10&times;10-3). The color scale is given on the bottom left corner of the image. The increase in fluorescence intensity for the chemical indicator-dosed beetle compared with the control beetle indicates that the oral dosing method successfully administers and delivers various chemical indicators in order to label the beetle leg muscle.&nbsp;Source: Graph from <strong>Oral Dosing of Chemical Indicators for In Vivo Monitoring of Ca<sup>2+</sup> Dynamics in Insect Muscle</strong> by Ferdinandus et al., <em>PLOS</em>, Jan. 2015.
Ca<sup>2+</sup> dynamics and muscle displacement of the beetle leg muscle under electrical stimulation with multiple pulse trains (100 Hz, 10% duty cycle, 2 V) for 3 s. Pseudocolor time series images of beetle leg muscle dosed with (A) Fluo-8 (60 &micro;M) and (C) Cell Tracker (60 &micro;M) indicators. ROIs are indicated by yellow region (as also shown in S1B Fig.). The color scale is given on the right side of each image (A) or (C) respectively. Fluorescence intensity dynamics of (B) Fluo-8 and (D) Cell Tracker under electrical stimulation, digitized with ImageJ software from the ROI shown in (A) and (C) respectively. The stimulus timing is indicated by grey shading. The Fluo-8 pseudocolor images illustrate the fluorescence intensity dynamics that correspond to the Ca<sup>2+</sup> dynamics inside the leg muscle: it increased at the start of electrical stimulation, was maintained during the application of the stimulus, and finally slowly decreased after the stimulus stopped. The Cell Tracker pseudocolor images display that the muscle displacement also causes intensity change during electrical stimulation, which slightly affects the Fluo-8 measurement. Source: Graph from <strong>Oral Dosing of Chemical Indicators for<em> In Vivo</em> Monitoring of Ca<sup>2+</sup> Dynamics in Insect Muscle</strong> by Ferdinandus et al., <em>PLOS,</em> Jan. 2015.
Relationship of Ca<sup>2+</sup> dynamics with electrical stimulation frequency. Relative changes in fluorescence intensity ((&Delta;F/F0)&times;100%) for leg muscle of (A) beetle orally dosed with Fluo-8 (blue) and Cell Tracker (red) and (B) control beetle measured with the filter setting used for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulations (1 Hz, 10 Hz, 50 Hz, and 100 Hz; 10% duty cycle; 2 V). Data were analyzed from the ROI adjacent to the stimulated site (S1B Fig.). The error bars represent the S.D. (N = 8 beetles, n = 24 beetle legs for (A); N = 2 beetles, n = 8 beetle legs for (B)). The small numbers next to each plot indicate the order of stimulation. Cell Tracker data set was compared with Fluo-8 data set at each stimulation frequency evaluated by student&rsquo;s t-test both for dosed beetles in (A) (1st 50 Hz, p = 9.37&times;10-4; 10 Hz, p = 7.45&times;10-3; 1st 1 Hz, p = 2.30&times;10-1; 100 Hz, p = 4.17&times;10-4; 2nd 1 Hz, p = 4.49&times;10-2; and 2nd 50 Hz, p = 8.16&times;10-3) and for control beetles in (B) (1st 50 Hz, p = 4.10&times;10-1; 10 Hz, p = 9.30&times;10-1; 1st 1 Hz, p = 9.29&times;10-1; 100 Hz, p = 5.69&times;10-2; 2nd 1 Hz, p = 6.85&times;10-1; and 2nd 50 Hz, p = 2.67&times;10-2). The significant differences are displayed by an asterisk (p &lt; 0.05). Fluo-8 intensity dynamics show that Ca<sup>2+</sup> dynamics inside the muscle have a positive correlation with electrical stimulation frequency; i.e., higher stimulation frequency induces larger increase in [Ca<sup>2+</sup>]. On the other hand, Cell Tracker intensity dynamics show that the frequency-dependent intensity change due to muscle displacement is not apparent. Source: Graph from <strong>Oral Dosing of Chemical Indicators for <em>In Vivo</em> Monitoring of Ca<sup>2+</sup> Dynamics in Insect Muscle</strong> by Ferdinandus et al., <em>PLOS</em>, Jan. 2015.
Effect of various electrical stimulation frequencies on Ca<sup>2+</sup> dynamics and muscle displacement in beetle leg muscle. Images of beetle leg muscle that was dosed with (A) Fluo-8 (60 &micro;M) and (C) Cell Tracker (60 &micro;M), with the yellow selection indicating the ROI that was used for analysis (as also shown in S1B Fig.). The color scale is given at the bottom left corner of the image. Representative time courses showing the fluorescence intensity dynamics of (B) Fluo-8, and (D) Cell Tracker under various electrical stimulations of multiple pulse trains (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V) observed from the ROI that are displayed in (A) and (C) respectively. All electrical stimulations were applied for 3 seconds periods with a 27 seconds resting period in between stimulations. The stimulus timing is indicated by grey shading. (E) Relative change in fluorescence intensity ((&Delta;F/F0)&times;100%) for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulation frequencies (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V). The small numbers next to each plot indicate the order of stimulation; i.e., first from 50 Hz followed by varying frequency pulses (10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz). The Fluo-8 intensity plot shows that Ca<sup>2+</sup> dynamics are dependent on electrical stimulation frequency, whereas the Cell Tracker plot shows that the muscle displacement contributes a small amount. Source: Graph from <strong>Oral Dosing of Chemical Indicators for <em>In Vivo</em> Monitoring of Ca<sup>2+</sup> Dynamics in Insect Muscle</strong> by Ferdinandus et al., <em>PLOS</em>, Jan. 2015.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)389
Molecular weight1046.93
SolventDMSO
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
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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Show More (66)

OverviewpdfSDSpdfProtocol


CAS
1345980-40-6
Molecular weight
1046.93
Dissociation constant (Kd, nM)
389
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 yield
0.161
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.

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

PREPARATION OF WORKING SOLUTION

Fluo-8® AM Working Solution
On the day of the experiment, either dissolve Fluo-8® 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, Fluo-8® 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-8® 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 Fluo-8® 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. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-8®, 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 mM95.517 µL477.587 µL955.174 µL4.776 mL9.552 mL
5 mM19.103 µL95.517 µL191.035 µL955.174 µL1.91 mL
10 mM9.552 µL47.759 µL95.517 µL477.587 µL955.174 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

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

Images


Citations


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References


View all 26 references: Citation Explorer
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Journal: Cell Calcium (2004): 509
Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux
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Journal: J Neurosci Methods (2004): 247
Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41
Authors: do Ceu Monteiro M, Sansonetty F, Goncalves MJ, O'Connor JE.
Journal: Cytometry (1999): 302
Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method
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Journal: Biophys J (1999): 606
A simple numerical model of calcium spark formation and detection in cardiac myocytes
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Journal: Shi Yan Sheng Wu Xue Bao (1998): 323
Loading and localization of Fluo-3 and Fluo-3/AM calcium indicators in sinapis alba root tissue
Authors: Tretyn A, Kado RT, Kendrick RE.
Journal: Folia Histochem Cytobiol (1997): 41
Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3
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Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites
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Journal: Pflugers Arch (1996): 893
Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets
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