Invitrogen™ Fluo-5F, AM, cell permeant - Special Packaging

Catalog No.	Quantity
F14222	10 x 50 μg

Catalog No. F14222 Supplier Invitrogen™ Supplier No. F14222

Description

Analog of fluo-4 with lower Ca²⁺-binding affinity, making it suitable for detecting intracellular calcium levels in 1μM to 1 mM range that would saturate response of fluo-3 and fluo-4

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca²⁺. Fluo-5F, fluo-5N, and fluo-4ff are analogs of fluo-4 with lower Ca²⁺-binding affinity, making them suitable for detecting intracellular calcium levels in the 1 μM to 1 mM range that would saturate the response of fluo-3 and fluo-4. 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 compatible with excitation at 488 nm by argon-ion laser sources, making them useful for confocal microscopy, flow cytometry, and microplate screening applications.

Calcium Indicator (AM Ester) Specifications:

• Label (Ex/Em of Ca²⁺–bound form): Fluo-5F (494/516 nm)
• Fluorescence intensity increase upon binding Ca²⁺: >100 fold
• K_d for Ca²⁺ in buffer: ∼2.3 μM
• 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 (Cat. No. TPEN).

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.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Order Info

Shipping Condition: Room temperature

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
• For Use With (Equipment): Confocal Microscope, Fluorescence Microscope, High Content Analysis Instrument, HTS Reader, Microplate Reader, Fluorescent Imager
• Product Type: Calcium Indicator
• Dye Type: Fluo-5F
• Quantity: 10 x 50 μg
• Shipping Condition: Room Temperature
• Excitation Wavelength: 494 nm
• Emission Wavelength: 516 nm

Frequently Asked Questions (FAQs)

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.

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