Cell-Based Ion Channel Assays - Labelilng & Detection

Fluo-4FF, AM, cell permeant - Special Packaging Invitrogen™

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-4ff (494/516 nm)
• Fluorescence intensity increase upon binding Ca2+: >100 fold
• Kd for Ca2+ in buffer: ~9.7 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 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.

Fura-2 Calcium Imaging Calibration Kit (Zero to 10 mM CaEGTA, 50 µM Fura-2) Invitrogen™

The Fura-2 Calcium Imaging Calibration Kit is designed to facilitate rapid calibration and standardization of digital imaging microscopes. The kit contains 11 pre-diluted buffers supplemented with 50 µM fura-2 (F-1200) as well as 15 µm—diameter microspheres.

BCECF, AM (2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) Invitrogen™

With a pKa of ~6.98, the cell-permeant, dual-excitation ratiometric pH indicator BCECF, AM is ideal for measuring changes in the cytosolic pH of most cells. Intracellular pH measurements with BCECF are made by determining the pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at ~490 nm versus the emission intensity when excited at its isobestic point of ~440 nm.

Premo™ Halide Sensor Invitrogen™

The Premo™ Halide Sensor is based on a yellow fluorescent protein (YFP) molecule sensitive to halide ions. The combination of the YFP (Venus) biosensor with efficient and non-cytopathic BacMam delivery and expression gives researchers a highly sensitive, robust and easy-to-use tool to efficiently screen halide ion channels and transporter modulators in their cellular models of choice. No specialized instruments are needed, as the sensor works on standard HTS platforms. - Cl– channel–specific—measure halide ion flux in voltage- and ligand-gated chloride channels and transporters - Fast—measure chloride flux in high-throughput mode with highly reproducible results - Robust—reliably high expression of the bright and fast maturating Venus sensor gives an excellent signal window - Convenient—study chloride flux in your cellular model by efficient and non-cytopathic delivery of Premo™ Halide Sensor by BacMam technology - Pharmacologically relevant—known modulators show dose-dependent quenching and BacMam delivery enables assays in primary cells The assay combines the YFP Venus halide sensor with a surrogate ion for chloride (iodide); upon stimulation of the chloride channel or transporter, iodide ions flow down the concentration gradient into the cells and quench YFP fluorescence upon binding; the amount of quench is directly proportional to the ion flux (chloride channel or transporter activity). Due to the bright Venus fluorescence, the Premo™ Halide Sensor signal window is excellent. We have demonstrated the utility and efficiency of the Premo™ Halide Sensor as a robust and true “no wash" HTS chloride assay. Premo™ Halide Sensor is pre-packaged and ready for immediate use. It contains all components required for cellular delivery and expression. Screening can be conducted in complete medium and without any wash steps. To uncouple cell maintenance and preparation from screening, simply freeze transduced cells and bring out as little as 4 h before the screen. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready".

Coelenterazine Invitrogen™

Coelenterazine and synthetic coelenterazine analogs are useful for reconstituting aequorin in cells that have been transfected with apoaequorin cDNA.

Fura-2, AM, cell permeant Invitrogen™

Fura-2, AM is a high affinity, intracellular calcium indicator that is ratiometric and UV light—excitable. This acetoxymethyl (AM) ester form is useful for noninvasive intracellular loading and is also available in 1 mg amounts (F-1201) and in a DMSO solution (F-1225). Cell-impermeant pentapotassium (F-1200) and pentasodium (F-6799) salt forms of the dye are available.

Fura-2, Pentasodium Salt, cell impermeant Invitrogen™

Fura-2, pentasodium salt is an intracellular calcium indicator that is ratiometric and UV light—excitable. This water-soluble salt form is useful for intracellular loading by microinjection, infusion from patch pipette or uptake induced by our Influx pinocytic cell-loading reagent (I-14402).

Rhod-3 Calcium Imaging Kit Invitrogen™

The novel Rhod-3 Calcium Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green-fluorescent dyes or proteins.The red-fluorescent Rhod-3 AM dye supplied in the Rhod-3 Calcium Imaging Kit offers significant improvements over existing Ca2+-sensing dyes. The uptake into organelles has been reduced, which translates into better cytosolic localization. Also, Rhod-3 AM exhibits a large increase in fluorescence emission upon binding to calcium, making it more sensitive than other red-fluorescent calcium dyes.

• Compared to existing red calcium dyes, Rhod-3 AM exhibits a large increase (>2.5 fold) in fluorescence upon binding Ca2+ and very low fluorescence at rest (without Ca2+ binding).

• Narrow emission enables simultaneous detection with GFP or other dyes.

• Enhanced cytosolic localization for better detection of cytosolic transients

For easy cell loading, the Rhod-3 Calcium Imaging Kit contains PowerLoad™ Concentrate. Due to the unique nature of the PowerLoad™ solution, it can be used in the presence of complete culture media, thus reducing the negative effects of replacing media or loading in serum-free media.

A proprietary, water-soluble form of probenecid (which is commonly used to inhibit cellular transport and thus reduces the baseline signal) is also supplied with the Rhod-3 Calcium Imaging Kit.

Calcium indicators are important tools in signal transduction and cell-based pharmacological screening. In cells and tissues with blue or green autofluorescence, long-wavelength (i.e., red-shifted) calcium indicators provide a means to bypass this overlapping autofluorescence. Additionally, red-shifted calcium dyes are well suited for calcium imaging experiments multiplexed with green-fluorescent protein (GFP) or other green fluorescent dyes.

The Rhod-3 AM dye (included in the Rhod-3 Calcium Imaging Kit) is an improved red-shifted rhodamine-based calcium dye with an attached AM ester group. Once inside the cell, intracellular esterases cleave the AM group, trapping the dye in the cell and rendering the dye fluorescent. As calcium binds, the fluorescence intensity of the dye is enhanced. The spectral properties of Rhod-3 AM (ex/em = ~560/600 nm) and strong fluorescence upon calcium binding provide an excellent signal window and make it our brightest red calcium dye. Additionally, Rhod-3 AM displays a more uniform cytosolic distribution and improved signal compared to existing red calcium dyes such as Rhod-2.

Fluo-4, AM, cell permeant Invitrogen™

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.

Fluo-3, AM, Calcium Indicator Invitrogen™

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-3 (506/526 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.

Fluo-3, AM, FluoroPure™ grade - Special Packaging Invitrogen™

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-3 (506/526 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.

PowerLoad™ Concentrate, 100X Invitrogen™

PowerLoad™ is an optimized formulation of nonionic, Pluronic® surfactant polyols for the solubilization of water-insoluble dyes and other materials in physiological media. These surfactants, for instance Pluronic® F-127, have been used to help disperse acetoxymethyl (AM) esters of fluorescent ion indicators such as fluo-4, fura-2, indo-1, fluo-3, and SBFI; they appear to be required for loading of other dyes (e.g. SBFI-AM or PBFI-AM). The use of PowerLoad™ is optional with red shifted calcium indicators and other large molecular weight AM ester dyes, and may also be useful for dispersing other lipophilic probes. The concentration of Pluronic® surfactants in PowerLoad™ is less than 0.2%. PowerLoad™ is effective in combination with water soluble Probenecid (P36400) to aid AM ester dye-loading and retention in cells that actively extrude the de-acetylated form through anion pumps. Together, these reagents allow for maximal loading of dyes with a minimum of effort in both imaging and high throughput screening (HTS) applications. Appropriate controls should be performed to make certain that PowerLoad™ is not altering the membrane properties of the cell.

Fluo-4 AM - Packaged For High-Throughput Screening Invitrogen™

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.

Fluo-4 NW Calcium Assay Kit Invitrogen™

Fluo-4 NW (No-Wash) Calcium Assay Kitsoffer a proprietary calcium assay formulation that requires neither a washstep nor a quencher dye. The fluo-4 NW assay achieves largerincreases in fluorescence intensity than standard fluo-3 and fluo-4assays with a wash step. Eliminating the wash step results inlower variability and higher Z´ values than the standard fluo-4 assay,while providing an easier and faster assay as well.

The fluo-4NW indicator is nonfluorescent and stable in pH 7–7.5 buffer forseveral hours, so spontaneous conversion to the Ca2+-sensitive formis not a significant source of background fluorescence. Contributionsto baseline fluorescence by the growth medium (e.g., esteraseactivity, proteins interacting with receptors of interest, or phenolred) are eliminated by removing the medium prior to adding theindicator dye to the wells.

Another source of potential fluorescenceoutside the cells is extrusion of the indicator out of the cell by organicanion transporters. Probenecid is commonly used to inhibitthis transport and reduce the baseline signal. We have synthesizeda proprietary water-soluble probenecid, which is supplied with theFluo-4 NW Calcium Assay Kits. This form of probenecid has theadvantages of being easy to dissolve in buffer and safer to use thanthe free acid, which requires caustic 1 M NaOH to dissolve. TheFluo-4 NW Calcium Assay Kits are designed for microplates andHTS, and the assay can be performed on adherent as well as nonadherentcells.

Fluo-4 AM is a fluorescent Ca+2 indicator that is widely usedfor in-cell measurement of agonist-stimulated and antagonist inhibitedcalcium signaling in high-throughput screening (HTS)applications. Its visible wavelength excitation (compatible withargon-ion laser sources), high sensitivity, and large fluorescenceincrease upon binding Ca2+ has made it the indicator of choicefor characterizing G-protein–coupled receptor (GPCR) pharmacologyand function. These properties have made fluo-4 AMattractive not only for microplate screening applications but formicroscopy and flow cytometry as well.

Fura-2, AM, cell permeant (1 mM Solution in Anhydrous DMSO) Invitrogen™

Fura-2, AM is a high affinity, intracellular calcium indicator that is ratiometric and UV light—excitable. This acetoxymethyl (AM) ester form is useful for noninvasive intracellular loading and is also available in 1 mg amounts (F-1201) and in special packagaing (F-1221). Cell-impermeant pentapotassium (F-1200) and pentasodium (F-6799) salt forms of the dye are available.
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