Molecular Probes™ Fluo-4, AM, cell permeant

Product Code.	Quantity	unitSize
11504786	10 x 50 μg	500µg

Product Code. 11504786 Supplier Molecular Probes™ Supplier No. F14201

Description

Fluo-4 is an analog of fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels.

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca²⁺. Fluo-3 has been used to image the spatial dynamics of Ca²⁺ signaling, in flow cytometry experiments involving photoactivation of caged chelators, second messengers, and neurotransmitters, and for cell-based pharmacological screening. Fluo-4 is an analog of fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. These indicators are useful for fluorescence and confocal microscopy, flow cytometry, and microplate screening applications.

Calcium Indicator (AM Ester) Specifications:

• Label (Ex/Em of Ca²⁺–bound form): Fluo-4 (494/506 nm)
• Fluorescence intensity increase upon binding Ca²⁺: >100 fold
• K_d for Ca²⁺ in buffer: ∼335 nM
• Exhibit fluorescence increase upon binding Ca²⁺ with little shift in wavelength

Using TPEN to Control Heavy Metal Cations

In addition, BAPTA-based indicators such as these bind various heavy metal cations (e.g., Mn²⁺, Zn²⁺, Pb²⁺) with substantially higher affinity than Ca²⁺. Perturbations to calcium measurements caused by presence of these ions can be controlled using the heavy metal-selective chelator TPEN (Cat. No. T1210).

More Choices for Fluorescent Calcium Indicators

We offer a large selection of Molecular Probes™ calcium indicators for use in various experimental scenarios. For more information, review Fluorescent Ca²⁺ Indicators Excited with Visible Light—Section 19.3 in the Molecular Probes™ Handbook.

For UV-excitable Ca²⁺ indicators, protein-based Ca²⁺ indicators, conjugates of Ca²⁺ indicators, and for fluorescence-based indicators of other metal ions (i.e., Mg²⁺, Zn²⁺) review Indicators for Ca²⁺, Mg²⁺, Zn²⁺ and Other Metal Ions—Chapter 19 in the Molecular Probes™ Handbook.

In reduced state, the excitation and emission maxima of BODIPY™ 581/591 undecanoic acid is 581/591 nm; after oxidation, the probe shifts the excitation and emission to 488/510 nm.

We recommend dissolving in high-quality anhydrous DMSO for stock concentrations of 1 to 5 mM.

Specifications

• Content And Storage: Store in freezer (-5°C to -30°C) and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Calcium Indicator, Cell Proliferation, Cellular Imaging
• For Use With (Equipment): Confocal Microscope, Fluorescence Microscope, High Content Analysis Instrument, HTS Reader, Microplate Reader, Fluorescent Imager
• Product Type: Dye
• Dye Type: Fluorescent Dye-Based
• Quantity: 10 x 50 μg
• Shipping Condition: Room Temperature
• Excitation/Emission: 494/506 nm

Frequently Asked Questions (FAQs)

Can I fix my cells after loading with Fluo-4 AM dye for the detection of calcium?

No. Since Fluo-4 AM isn't covalently bound to any cellular components and fixation compromises the membrane, the dye would not be retained by the cell.

I am doing calcium flux imaging with your Fura-2 calibration kit, but am seeing a large variability in ratio in different places around the slide. I am correcting for uniform illumination, using the product as directed, and sealing the coverslip with nail polish.

The nail polish may be the problem. The K_d value (calcium sensitivity) changes depending upon the dye's environment. Nail polish has solvents that can leech under the coverslip and cause variability. We recommend either going without a sealing or sealing with melted paraffin painted on the coverslip edges with a cotton-tipped applicator (paraffin is hydrophobic and has no solvents).

I need to label cells with Fluo-4, AM, for a calcium flux assay. How long after labeling will the dye be retained?

After loading dye into the cells, intracellular esterases remove the 'AM' moiety from the dye. When the 'AM' group is removed, the dye is able to bind calcium and fluoresce. Since the dye is not covalently bound to any cellular components, it may be actively effluxed from the cell. The rate of efflux is dependent upon the inherent properties of the cell, culture conditions and other factors. The dye may be retained for hours, days or even weeks or lost in a matter of minutes. The use of Probenecid (Cat. No. P36400) limits loss by active efflux.

Why don't I see a significant change in signal for my live-cell fluorescent indicator dye?

Regardless of the type of live-cell indicator dye (e.g., calcium indicators, pH indicator, metal ion indicators), make sure there is no serum during the loading step, which can prematurely cleave dyes with AM esters and bind dyes non-specifically. Always optimize the dye concentration and staining time with a positive control before you run your test samples, to give the best signal-to-background. Always run a positive control with a buffer containing free ions of known concentration and an ionophore to open pores to those ions (for instance, for calcium indicators like Fluo-4 AM, this would include a buffer with added calcium combined with calcimycin, or for pH indicators, buffers of different pHs combined with nigericin). Reactive oxygen indicators, such as CellROX Green or H2DCFDA would require a cellular reactive oxygen species (ROS) stimulant as a positive control, such as menadione. Finally, make sure your imaging system has a sensitive detector. Plate readers, for instance, have much lower detector efficiency over background, compared to microscopy or flow cytometry.

For Research Use Only. Not for use in diagnostic procedures.