Fluo-8H™, 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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Additional ordering information

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

Physical properties

Dissociation constant (K_d, nM)	232
Molecular weight	1074.98
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

1074.98

Dissociation constant (K_d, nM)

232

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-8H™ AM Stock Solution

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

PREPARATION OF WORKING SOLUTION

Fluo-8H™ AM Working Solution

On the day of the experiment, either dissolve Fluo-8H™ 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-8H™ 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-8H™ 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-8H™ 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-8H™, 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	93.025 µL	465.125 µL	930.25 µL	4.651 mL	9.302 mL
5 mM	18.605 µL	93.025 µL	186.05 µL	930.25 µL	1.86 mL
10 mM	9.302 µL	46.512 µL	93.025 µL	465.125 µL	930.25 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

Product Family

Name	Excitation (nm)	Emission (nm)	Quantum yield
Fluo-8®, AM	495	516	0.16¹
Fluo-8L™, AM	495	516	0.16¹
Fluo-8FF™, AM	495	516	0.16¹
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	0.16¹
Fluo-3, AM CAS 121714-22-5	507	516	0.15¹
Fluo-3, AM UltraPure grade CAS 121714-22-5	507	516	0.15¹
Fluo-3, AM Bulk package CAS 121714-22-5	507	516	0.15¹
Fluo-3FF, AM UltraPure grade Cell permeant	507	516	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

View all 172 citations: Citation Explorer

The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis
Authors: Shiba, Kogiku and Inaba, Kazuo
Journal: International Journal of Molecular Sciences (2022): 1648

Crosstalk between ENaC and basolateral Kir4. 1/Kir5. 1 channels in the cortical collecting duct
Authors: Isaeva, Elena and Bohovyk, Ruslan and Fedoriuk, Mykhailo and Shalygin, Alexey and Klemens, Christine A and Zietara, Adrian and Levchenko, Vladislav and Denton, Jerod S and Staruschenko, Alexander and Palygin, Oleg
Journal: British journal of pharmacology (2021)

Correlation between calcium signaling and ATP release mediated by connexin hemichannels in different mouse models of disease
Authors: Mazzarda, Flavia
Journal: (2021)

Development of vibration mechanical stimuli loading device for live cell fluorescence microscopy
Authors: SATO, Katsuya and OOMORI, Daiki
Journal: Journal of Biomechanical Science and Engineering (2021): 21--00294

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca 2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea
Authors: Mazzarda, Flavia and D'Elia, Annunziata and Massari, Roberto and De Ninno, Adele and Bertani, Francesca Romana and Businaro, Luca and Ziraldo, Gaia and Zorzi, Veronica and Nardin, Chiara and Peres, Chiara and others,
Journal: Lab on a Chip (2020): 3011--3023

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens
Authors: Makino, Nanae and Sato, Nozomi and Takayama-Watanabe, Eriko and Watanabe, Akihiko
Journal: Reproduction (2020): 339--349

A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites
Authors: Bhandage, Amol K and Olivera, Gabriela C and Kanatani, Sachie and Thompson, Elizabeth and Lor{\'e}, Karin and Varas-Godoy, Manuel and Barragan, Antonio
Journal: Elife (2020): e60528

A Distinct Role of the Autonomic Nervous System in Modulating the Function of Lymphatic Vessels under Physiological and Tumor-Draining Conditions
Authors: Bachmann, Samia B and Gsponer, Denise and Montoya-Zegarra, Javier A and Schneider, Martin and Scholkmann, Felix and Tacconi, Carlotta and Noerrelykke, Simon F and Proulx, Steven T and Detmar, Michael
Journal: Cell reports (2019): 3305--3314

Droplet microfluidic systems for formation and studies of lipid bilayers
Authors: Czekalska, Magdalena Anna
Journal: (2019)

Electrode-free nanopore sensing by DiffusiOptoPhysiology
Authors: Wang, Yuqin and Wang, Yu and Du, Xiaoyu and Yan, Shuanghong and Zhang, Panke and Chen, Hong-Yuan and Huang, Shuo
Journal: Science Advances (2019): eaar3309