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.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

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Physical properties

Dissociation constant (K_d, nM)	1900
Molecular weight	1078.95
Solvent	DMSO

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

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

1078.95

Dissociation constant (K_d, 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
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Spectrum

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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 yield	0.16¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield
Fluo-8®, AM	495	516	23430	0.16¹
Fluo-8H™, AM	495	516	23430	0.16¹
Fluo-8FF™, AM	495	516	23430	0.16¹
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	82000	0.16¹
Fluo-3, AM CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3, AM UltraPure grade CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3, AM Bulk package CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3FF, AM UltraPure grade Cell permeant	506	515	86,000¹	0.15¹
Fluo-5F, AM Cell permeant	494	516	-	-
Fluo-5N, AM Cell permeant	494	516	-	-
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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

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Journal: (2023)

Smooth muscle AKG/OXGR1 signaling regulates epididymal fluid acid-base balance and sperm maturation
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Journal: Life Metabolism (2022)

The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria
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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

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)

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 βTC-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