Cell-Based Second Messenger Assays - Indicators

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.

Fluo-4, 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-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-4FF, Pentapotassium Salt, cell impermeant (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 physically loaded with the cell-impermeant salt forms of these indicators using patch pipette or microinjection or our Influx™ pinocytotic cell-loading reagent. 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 (Cell-Impermeant Salts) 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.

Oregon Green™ 488 BAPTA-1, AM, cell permeant - Special Packaging (Invitrogen™)

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca2+. They have uses in many calcium signaling investigations, including measuring intracellular Ca2+, following Ca2+ influx and release, and multiphoton excitation imaging of Ca2+ in living tissues. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. The fluorescence signal from these cells is generally measured using fluorescence microscopy, fluorescence microplate assays, or flow cytometry.

Calcium Indicator (AM Ester) Specifications:
• Label (Ex/Em): Oregon Green® 488 BAPTA-1 (494/523 nm)
• Fluorescence intensity increase upon binding Ca2+ : ~14 fold
• Exhibit fluorescence increase upon binding Ca2+ with little shift in wavelength


Spectral Characteristics of Molecular Probes® Calcium Indicators
These probes are excited by visible light, and because the energy required for excitation is low, the potential for cellular photodamage is reduced. Commonly used laser-based instruments (i.e., confocal laser scanning microscopes) are able to efficiently excite these indicators, and their emissions are in regions of the spectrum where cellular autofluorescence and scattering backgrounds are often less of a problem.

More Choices for Fluorescent Calcium Indicators
We offer a large selection of Molecular Probes® calcium indicators for use in various experimental scenarios, for example dextran versions for reduced leakage and compartmentalization and BAPTA conjugates for detecting high-amplitude calcium transients. 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 intended for animal or human therapeutic or diagnostic use.

cAMP-Screen™ Cyclic AMP Immunoassay System (Applied Biosystems™)

The cAMP-Screen® 96-well Cyclic AMP Immunoassay System enables ultrasensitive determination of cyclic AMP (cAMP) levels in cell lysates. This competitive chemiluminescent immunoassay is formatted with maximum flexibility to permit either manual reagent additions or automated high-throughput processing. The cAMP assay utilizes the highly sensitive chemiluminescent CSPD® Substrate with Sapphire-II™ Enhancer that is triggered by an enzyme conjugate composed of cAMP bound to alkaline phosphatase (cAMP-AP). The chemiluminescent substrate/enhancer formulation is a ready-to-use reagent that generates a sustained-glow light emission from 30 minutes after addition. This assay is mainly used in secondary screening and pre-clinical research, where sensitivity and no false positives are essential.

• The highest sensitivity of any commercially available cAMP assay
• Hours of read time with little or no degradation of the signal
• A simple protocol
• Useful for a wide range of receptor activation studies

High Sensitivity and Hours of Steady Glow Time
This chemiluminescent assay is designed to provide the highest sensitivity of any commercially available cAMP assay. As few as 60 femtomoles of cAMP can be detected. The assay has a wide dynamic range, detecting from 0.06 to 6,000 picomoles of cAMP without the need for sample dilution or manipulations such as acetylation. This is especially helpful in cell-based assays when measuring Gs- or Gi-coupled agonist or antagonist stimulation and/or inhibition. Intra-assay precision for duplicate samples is typically 5% or less. Once the substrate/enhancer formulation reaches glow signal, the plate can be read for hours with little or no degradation of the signal. This is useful in screens where several plates are compared with each other. In addition, the assay exhibits exceptionally low cross-reactivity with other adenosine-containing or cyclic nucleotides.

For Receptor Activation Studies
The cAMP-Screen® system is designed for quantitation of cellular cAMP for functional assays of receptor activation. The assay has been used with established cell lines for functional measurements with endogenous receptors, cell lines with exogenously expressed ligand receptors on the cell surface, primary cell cultures, and tissues in response to treatment with the appropriate ligands. In addition it has been used for receptor characterization, orphan receptor ligand identification, and the characterization of novel chimeric receptors. The assay can be used for high-throughput screens for compounds that stimulate or interfere with these signal transduction pathways.

A Simple Protocol
The assay follows a simple protocol. Cells are seeded into plates, cultured, and treated with test compounds as desired. Cell lysates are prepared in either the presence or absence of culture media. Lysates are incubated with a cAMP-AP conjugate and an anti-cAMP antibody in a coated microplate; the resulting immune complexes are captured in the plate. In samples without cAMP, all of the cAMP-AP conjugate is captured on the coated surface, resulting in a high signal. In the presence of cAMP, the amount of cAMP-AP conjugate captured decreases as a result of competition for binding with unlabeled cAMP, causing a reduced signal; signal reduction is proportional to the amount of cAMP present in the cell lysate. After washing to remove unbound cAMP-AP, the chemiluminescent substrate is added, and the resulting glow signal is measured in a luminometer.

An alternate product, the cAMP-Screen Direct® system, has all of the same benefits as this cAMP-Screen® system, but eliminates the need to transfer cell lysates from a tissue culture plate to the precoated microplates because the cells can be grown directly in the microplates. This provides better assay precision with one less transfer step. Many different cell types have been grown successfully on the precoated microplates, which also have a clear bottom to allow monitoring of cells.

For Research Use Only. 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.

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, 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).

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.

Fura Red™, AM, cell permeant (Invitrogen™)

Fura Red, AM is a visible light—excitable fura-2 analog that offers unique possibilities for ratiometric measurement of Ca2+ in single cells by microphotometry, imaging or flow cytometry when used with single excitation, green-fluorescent calcium indicators. This acetoxymethyl (AM) ester form is useful for noninvasive intracellular loading and is also available in 1 mg amounts (F-3020).

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 special packaging (F-1221) and in a DMSO solution (F-1225). Cell-impermeant pentapotassium (F-1200) and pentasodium (F-6799) salt forms of the dye are available.

Fluo-3, Pentaammonium Salt, cell impermeant (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 physically loaded with the cell-impermeant salt forms of these indicators using patch pipette, microinjection, or our Influx™ pinocytotic cell-loading reagent. These indicators are useful for fluorescence and confocal microscopy, flow cytometry, and microplate screening applications.

Calcium Indicator (Cell-Impermeant Salts) 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-4, Pentapotassium Salt, cell impermeant (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 physically loaded with the cell-impermeant salt forms of these indicators using patch pipette, microinjection, or our Influx™ pinocytotic cell-loading reagent. These indicators are useful for fluorescence and confocal microscopy, flow cytometry, and microplate screening applications.

Calcium Indicator (Cell-Impermeant Salts) Specifications:
• Label (Ex/Em of Ca2+–bound form): Fluo-4 (494/516 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.

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.

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.