Fluo-8L™, AM
| Overview |  SDSProtocol |
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
| Excitation | FITC |
| Emission | FITC |
| Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
| Excitation | 490 |
| Emission | 525 |
| Cutoff | 515 |
| Recommended plate | Black 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
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
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.
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.
- Prepare cells in growth medium overnight.
- 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. - 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. - 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.
- 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 92.683 µL | 463.414 µL | 926.827 µL | 4.634 mL | 9.268 mL |
| 5 mM | 18.537 µL | 92.683 µL | 185.365 µL | 926.827 µL | 1.854 mL |
| 10 mM | 9.268 µL | 46.341 µL | 92.683 µL | 463.414 µL | 926.827 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Open in Advanced Spectrum Viewer
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 yield | 0.161 |
Images
Figure 1. 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.
Citations
View all 156 citations: Citation Explorer
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
Authors: Phillips, Wiktor S and Del Negro, Christopher A and Rekling, Jens C
Journal: Journal of Neuroscience (2018): 3039--3049
Human lysophosphatidic acid receptor 2-K1. 31 and K7. 36 gatekeeper functions provide new insight into robust affinity for LPA-type agonists.
Authors: Omotuyi, Olaposi I
Journal: Journal of Systems Biology & Proteome Research (2017)
Authors: Omotuyi, Olaposi I
Journal: Journal of Systems Biology & Proteome Research (2017)
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
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
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
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 βTC-3 pancreatic cells: Evaluation of structure-dependent biological activity
Authors: Drzazga, Anna and Sowińska, Agata and Krzemińska, 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)
Authors: Drzazga, Anna and Sowińska, Agata and Krzemińska, 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
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
Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions
Authors: Thillaiappan, Nagendra Babu and Chavda, Alap P and Tovey, Stephen C and Prole, David L and Taylor, Colin W
Journal: Nature Communications (2017): 1505
Authors: Thillaiappan, Nagendra Babu and Chavda, Alap P and Tovey, Stephen C and Prole, David L and Taylor, Colin W
Journal: Nature Communications (2017): 1505
Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage
Authors: Gong, Xiaoyuan and Xie, Wenbin and Wang, Bin and Gu, Lingchuan and Wang, Fuyou and Ren, Xiang and Chen, Cheng and Yang, Liu
Journal: Scientific reports (2017): 17093
Authors: Gong, Xiaoyuan and Xie, Wenbin and Wang, Bin and Gu, Lingchuan and Wang, Fuyou and Ren, Xiang and Chen, Cheng and Yang, Liu
Journal: Scientific reports (2017): 17093
A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels
Authors: Menegon, A and Pitassi, S and Mazzocchi, N and Redaelli, L and Rizzetto, R and Roll, undefined and , JF and Poli, C and Imberti, M and Lanati, A and Grohovaz, F
Journal: Scientific Reports (2017)
Authors: Menegon, A and Pitassi, S and Mazzocchi, N and Redaelli, L and Rizzetto, R and Roll, undefined and , JF and Poli, C and Imberti, M and Lanati, A and Grohovaz, F
Journal: Scientific Reports (2017)
Application notes
Fluo-8® Calcium Reagents and Screen Quest™ Fluo-8 NW Calcium Assay Kits
Fluorescent Dye AM Esters
The Eight Best Green Fluorescent Calcium Indicators
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A highly sensitive fluorescent dye for calcium imaging of neural activity
FAQ
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