Fluo-8® Calcium Indicators

In this issue

Fluorescent Calcium Indicators
Fluo-8® Calcium Indicators
Cal-520™ Calcium Indicators
Cal-590™ Calcium Indicators
Cal-630™ Calcium Indicators
Rhod-4™ Calcium Indicators
Ratiometric Calcium Indicators
FLIPR® Calcium Assays

AssayWise Letters 2015, Vol. 4(1)
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Carbachol dose responses were measured in HEK-293 cells with Fluo-8<sup>®</sup> AM

Carbachol dose responses were measured in HEK-293 cells with Fluo-8^® AM (Cat# 21080) and Fluo-4 AM. HEK-293 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 100 µL of dye-loading solution containing Fluo-8^® AM or Fluo-4 AM for 1 hour at room temperature. Carbachol (25 µL/well) was added by NOVOstar to achieve the final indicated concentrations. The fluorescence signals were measured at Ex/Em = 490/525 nm. The EC50 of carbachol measured with Fluo-8^® AM is about 1.2 µM.

Fluo-3 and Fluo-4 were the most commonly used visible light-excitable calcium indicators. 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 have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of maximum excitation @ ~490 nm and maximum emission @ ~520 nm. For cell loading, Fluo-8^® AM only requires incubation at room temperature while Fluo-3 AM and Fluo-4 AM require incubation at 37 °C. In addition, Fluo-8^® AM is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM in cells. AAT Bioquest offers a set of out-standing Fluo-8^® reagents with different calcium binding affinities.

Key Features of Fluo-8^® AM:

Faster, more readily loaded into cells than Fluo-3 AM and Fluo-4 AM. Only room temperature is required.
Brighter, much brighter than Fluo-3 AM and Fluo-4 AM in cells.
Convenient, almost identical spectra to those of Fluo-4 AM.

U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of 4 µM Fluo-3 AM, Fluo-4 AM and Fluo-8^® AM (Cat# 21080) in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope using FITC channel.

Table 1. Fluo-8® green fluorescent calcium indicators for live cell calcium imaging.

Indicator ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Kd¹ ▲ ▼	Φ² ▲ ▼	FCa/FFree³ ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
Fluo-8®, AM	495	516	389 nM	0.16	∼200 fold	1 mg	21080
Fluo-8®, AM	495	516	389 nM	0.16	∼200 fold	10x50 µg	21082
Fluo-8®, AM	495	516	389 nM	0.16	∼200 fold	20x50 µg	21083
Fluo-8®, AM	495	516	389 nM	0.16	∼200 fold	5x50 µg	21081
Fluo-8H™, AM	495	516	232 nM	0.16	∼200 fold	10x50 µg	21091
Fluo-8H™, AM	495	516	232 nM	0.16	∼200 fold	1 mg	21090
Fluo-8L™, AM	495	516	1.9 µM	0.16	∼200 fold	1 mg	21096
Fluo-8L™, AM	495	516	1.9 µM	0.16	∼200 fold	10x50 µg	21097
Fluo-8FF™, AM	495	516	10 µM	0.16	∼200 fold	10x50 µg	21104
Fluo-8FF™, AM	495	516	10 µM	0.16	∼200 fold	1 mg	21105
Fluo-8®, potassium salt	495	516	389 nM	0.16	∼200 fold	1 mg	21087
Fluo-8®, potassium salt	495	516	389 nM	0.16	∼200 fold	10x50 µg	21089
Fluo-8®, sodium salt	495	516	389 nM	0.16	∼200 fold	1 mg	21086
Fluo-8®, sodium salt	495	516	389 nM	0.16	∼200 fold	10x50 µg	21088
Fluo-8H™, sodium salt	495	516	232 nM	0.16	∼200 fold	10x50 µg	21095
Fluo-8L™, sodium salt	495	516	1.9 µM	0.16	∼200 fold	10x50 µg	21098
Fluo-8L™, sodium salt	495	516	1.9 µM	0.16	∼200 fold	1 mg	21099

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Fluo-8® Calcium Indicators

In this issue

Key Features of Fluo-8® AM:

Table 1. Fluo-8® green fluorescent calcium indicators for live cell calcium imaging.

Key Features of Fluo-8^® AM: