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Molecular Probes™ Fluo-4 AM - Packaged For High-Throughput Screening

Catalog No. F14202
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Description

fluo-4 AM - packaged for high-throughput screening

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

  • Kd 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.

    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.

    • Specifications

    Content And Storage Store at -5°C to -30°C and protect from light.
    Detection Method Fluorescence
    For Use With (Application) Cell Viability, Proliferation and 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 5 x 1 mg
    Shipping Condition Room Temperature

    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 Kd 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.