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AAT Bioquest

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.
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.
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Physical properties
Dissociation constant (Kd, nM)1900
Molecular weight1078.95
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
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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OverviewpdfSDSpdfProtocol


Molecular weight
1078.95
Dissociation constant (Kd, nM)
1900
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-8L™ AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fluo-8L™ AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fluo-8L™ AM Working Solution
  1. 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.

  2. Prepare a 2 to 20 µM Fluo-8L™ 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-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 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-8L™ 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-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.

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

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-8®, AM495516234300.161
Fluo-8H™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528820000.161
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-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--
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Images


Citations


View all 161 citations: Citation Explorer
The role of OXGR1 on gut smooth muscle to regulate intestinal motility and health
Authors: Xu, Guli and Zhou, Jingjing and Gyawali, Ishwari and Feng, Jinlong and Yuan, Yexian and Xu, Chang and Yang, Jinping and Ma, Zewei and Li, Penglin and Sui, Chengrong and others,
Journal: (2023)
Smooth muscle AKG/OXGR1 signaling regulates epididymal fluid acid-base balance and sperm maturation
Authors: Xu, Chang and Yuan, Yexian and Zhang, Cha and Zhou, Yuchuan and Yang, Jinping and Yi, Huadong and Gyawali, Ishwari and Lu, Jingyi and Guo, Sile and Ji, Yunru and others,
Journal: Life Metabolism (2022)
The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria
Authors: Bassot, Arthur and Chen, Junsheng and Takahashi-Yamashiro, Kei and Yap, Megan C and Gibhardt, Christine Silvia and Le, Giang NT and Hario, Saaya and Nasu, Yusuke and Moore, Jack and Guti{\'e}rrez, Tomas and others,
Journal: Cell Reports (2022): 111899
Dendritic A-current in rhythmically active preb{\"o}tzinger complex neurons in organotypic cultures from newborn mice
Authors: Phillips, Wiktor S and Del Negro, Christopher A and Rekling, Jens C
Journal: Journal of Neuroscience (2018): 3039--3049
Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
Authors: Hirata, Yusuke and Morimoto, Yuya and Nam, Eunryel and Yoshida, Shotaro and Takeuchi, Shoji
Journal: (2017): 13--16
Z-360 Suppresses Tumor Growth in MIA PaCa-2-bearing Mice via Inhibition of Gastrin-induced Anti-Apoptotic Effects
Authors: SHIOMI, YOSHIHIRO and YOSHIMURA, MAKOTO and KUKI, KAZUMASA and HORI, YUKO and TANAKA, TAKAO
Journal: Anticancer Research (2017): 4127--4137
Laminarin counteracts diet-induced obesity associated with glucagon-like peptide-1 secretion
Authors: Yang, Liusong and Wang, Lina and Zhu, Canjun and Wu, Junguo and Yuan, Yexian and Yu, Lulu and Xu, Yaqiong and Xu, Jingren and Wang, Tao and Liao, Zhengrui and others, undefined
Journal: Oncotarget (2017): 99470
2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from &beta;TC-3 pancreatic cells: Evaluation of structure-dependent biological activity
Authors: Drzazga, Anna and Sowi&nacute;ska, Agata and Krzemi&nacute;ska, Agnieszka and Okruszek, Andrzej and Paneth, Piotr and Koziolkiewicz, Maria and Gendaszewska-Darmach, Edyta
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)
L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes
Authors: Wu, Jianjun and Wang, Xiangchong and Chung, Ying Ying and Koh, Cai Hong and Liu, Zhenfeng and Guo, Huicai and Yuan, Qiang and Wang, Chuan and Su, Suwen and Wei, Heming
Journal: PloS one (2017): e0168435