Cell-Based Ion Channel Assays - Labelilng & Detection

FluxOR™ Potassium Ion Channel Assay Invitrogen™

K+ channel specific—measure ion flux in both voltage- and ligand-gated potassium channels
Fast—perform screens in high-throughput mode with reproducible results and excellent S/N without quenching dye
Pharmacologically relevant—known blockers show dose-dependent inhibition in a large signal window

The FluxOR™ Potassium Ion Channel Assay is an optically based, homogenous assay for high throughput screening (HTS) measurements of potassium ion channel and transporter activities. The homogenous assay is based on the permeability of potassium channels to thallium I. When potassium channels are opened by a stimulus, thallium influx from the external medium is detected with a highly sensitive indicator dye. The fluorogenic signal quantitatively reflects the activity of ion channels and transporters that are permeant to thallium, including hERG, Kir2.1, and other pharmacologically important potassium channels. Fluorescence reported in the FluxOR™ system thus becomes a surrogate indicator of activity for any ion channel or transporter that is permeable to thallium.

The FluxOR™ Potassium Ion Channel Assay enables rapid and robust high-throughput screening (HTS) of potassium channel targets in a novel equilibrium measurement, reproducibly giving IC50 values that are predictive of block or modulation in lower-throughput platforms. The FluxOR™ dye is sensitive enough that low mM levels of extracellular thallium give large signals in high-throughput mode. For most applications, FluxOR™ dye is dissolved in physiological HBSS buffer for loading into cells, assisted by our proprietary Powerload™ formulation (Catalog number P10020).

The FluxOR™ Potassium Ion Channel Assay provides a concentrated thallium solution and all necessary buffers, allowing maximum target flexibility and ease of operation in a homogenous format that has been demonstrated for use with cells stably expressing hERG, as well as our BacMam-hERG delivery and expression reagent (Catalog number B10019 and B10033). Optional for use with this kit is the hERG potassium channel cDNA engineered into Invitrogen’s BacMam delivery and expression system. The combination of a bright proprietary fluorescent potassium sensor dye and the hERG potassium channel gene delivered by BacMam affords excellent assay design flexibility and superior sensitivity in detecting potassium channel activity in biologically relevant systems at physiological conditions without the need for quenchers.

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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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, 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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

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".

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

Probenecid, Water Soluble Invitrogen™

Probenecid is commonly used to inhibit organic-anion transporters located in the cell membrane. Such transporters can extrude dyes and indicators and thus contribute to poor loading or a high background signal in assays based on retention of the dyes or indicators inside cells. The use of probenecid to block the efflux of intracellular dyes was first described by Di Virgilio et al. (1990), and it has been used with a wide range of anionic dyes and conjugates. The commonly used free acid form of probenecid is difficult to dissolve, requiring 1 M NaOH to get it into solution. Our water-soluble probenecid (P36400) dissolves quickly in assay buffer and eliminates the need to handle caustic NaOH. The water-soluble probenecid is included in the Fluo-4 NW Calcium Assay Kits (F36205, F36206) and is also available separately.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

SBFI, AM, cell permeant - Special Packaging Invitrogen™

SBFI is a sodium-sensitive molecule used to estimate Na+ gradients in isolated mitochondria, to measure intracellular NA+ levels, to measure Na+ efflux in cells, and, in combination with other fluorescent indicators used to correlate changes in intracellular Na+ with Ca2+ and Mg2+ concentrations, intracellular pH, and membrane potential. Although the selectivity of SBFI for Na+ is less than that of calcium indicators such as fura-2, it is sufficient for the detection of physiological concentrations of Na+ in the presence of other monovalent cations. The spectral response of SBFI upon ion binding permit excitation ratio measurements, and this indicator can be used with the same optical filters and equipment used for fura-2.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

Fluorescent Ion Indicators Specifications:
• Label (Ex/Em): SBFI (~340,380/500 nm)
• Lyophilized product may be dissolved in DMSO for use
• Product is typically loaded into cells by adding the dissolved indicator to medium containing cells


Selectivity Considerations and Cell Loading Strategies
The dissociation constant (Kd) of SBFI for Na+ is 3.8 mM in the absence of K+, and 11.3 mM in solutions with a combined Na+ and K+ concentration of 135 mM (which approximates physiological ionic strength). SBFI is ~18-fold more selective for Na+ than for K+.

SBFI is available as both cell-impermeant acid salt (S1262) and as cell-permeant acetoxymethyl (AM) esters (S1263, S1264). The anionic acid forms can be loaded into cells using our Influx™ pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), or by microinjection, patch-pipette infusion or electroporation. For AM ester loading (Loading and Calibration of Intracellular Ion Indicators—Note 19.1), addition of the Pluronic® F-127 (P3000MP, P6866, P6867) or PowerLoad™ (P10020) dispersing agents as well as relatively long incubation times—up to four hours—are typically necessary.

Find Fluorescent Indicators for Na+ and K+
We offer a number of fluorescent indicators for measuring Na+ and K+. Review Fluorescent Na+ and K+ Indicators—Section 21.1 in the Molecular Probes® Handbook for more information on these products.

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

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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

PBFI, AM, cell permeant - Special Packaging Invitrogen™

PBFI is a potassium-sensitive molecule used measure intracellular K+ fluxes in animal cells and in plant cells and vacuoles. Although the selectivity of PBFI for K+ is less than that of calcium indicators such as fura-2, it is sufficient for the detection of physiological concentrations of K+ in the presence of other monovalent cations. The spectral response of PBFI upon ion binding permit excitation ratio measurements, and this indicator can be used with the same optical filters and equipment used for fura-2.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

Fluorescent Ion Indicators Specifications:
• Label (Ex/Em): PBFI (~340,380/500 nm)
• Lyophilized product may be dissolved in DMSO for use
• Product is typically loaded into cells by adding the dissolved indicator to medium containing cells


Selectivity Considerations and Cell Loading Strategies
The Kd of PBFI for K+ is strongly dependent on whether Na+ is present, with a value of 5.1 mM in the absence of Na+ and 44 mM in solutions with a combined Na+ and K+ concentration of 135 mM (which approximates physiological ionic strength). In buffers in which the Na+ is replaced by tetramethylammonium chloride, the Kd of PBFI for K+ is 11 mM; choline chloride and N-methylglucamine are two other possible replacements for Na+ in the medium. Although PBFI is only 1.5-fold more selective for K+ than for Na+, this selectivity is often sufficient because intracellular K+ concentrations are normally about 10 times higher than Na+ concentrations.

PBFI is available as both cell-impermeant acid salt (P1265MP) and as cell-permeant acetoxymethyl (AM) esters (P1267MP). The anionic acid forms can be loaded into cells using our Influx™ pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), or by microinjection, patch-pipette infusion or electroporation. For AM ester loading (Loading and Calibration of Intracellular Ion Indicators—Note 19.1), addition of the Pluronic® F-127 (P3000MP, P6866, P6867) or PowerLoad™ (P10020) dispersing agents as well as relatively long incubation times—up to four hours—are typically necessary.

Find Fluorescent Indicators for Na+ and K+
We offer a number of fluorescent indicators for measuring Na+ and K+. Review Fluorescent Na+ and K+ Indicators—Section 21.1 in the Molecular Probes® Handbook for more information on these products.

For Research Use. 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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

Indo-1, AM, cell permeant - Special Packaging Invitrogen™

This cell-permeant indo-1, AM is a UV light—excitable, ratiometric Ca2+ indicator. The emission maximum of indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca2+. This AM ester form of the dye is available in 1mg amounts (I-1203) and in 1 mM solution (I-1226). A cell-impermeant pentapotassium (F-1202) salt form of the dye is also available.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

Fura-2, AM, FluoroPure™ grade - Special Packaging Invitrogen™

Because it is manufactured at our ISO 9001—certified facilities in Eugene, Oregon, we can guarantee that FluoroPure grade Fura-2 AM is greater than or equal to 98% pure by HPLC.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

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.
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