Cell-Based Second Messenger Assays - Indicators

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-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.

Indo-1, AM, cell permeant (Invitrogen™)

Indo-1, AM is a high affinity, intracellular calcium indicator (Kd ~0.23 µM) 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 (I-1223) and in a DMSO solution (I-1226).

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.

Fluo-4 Direct™ Calcium Assay Kit (Invitrogen™)

The Fluo-4 Direct™ Calcium Assay Kit was formulated to provide a homogeneous fluorescent calcium assay that:
1. Easily loads into cells
2. Achieves a large assay window and
3. Suppresses background fluorescence generated from the calcium indicator in complete media with little-to-no impact on the specific cellular fluorescence

This advanced formulation allows the assay to be run in a simple “addition only" format in the presence of serum-containing media. Fluo-4 Direct™ is the third addition to the Fluo-4 family of calcium detection reagents. Fluo-4 AM and Fluo-4 NW both require media removal before assay detection, but Fluo-4 NW adds convenience by including the PowerLoad™ reagent for ease in cell loading. The Fluo-4 Direct™ Calcium Assay Kit is similar to Fluo-4 NW in that it is formulated with PowerLoad™ for ease in cell loading, but unique in that it can be used in the presence of complete culture media and will efficiently suppress background fluorescence without sacrificing the specific cellular fluorescence generated in the assay.

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-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.

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-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.

Indo-1, AM, cell permeant (Invitrogen™)

Indo-1, AM is a high affinity, intracellular calcium indicator (Kd ~0.23 µM) that is ratiometric and UV light—excitable. This acetoxymethyl (AM) ester form is useful for noninvasive intracellular loading and is also available in a 1 mg packaging (I-1203) and a special packaging (I-1223).

Coelenterazine h (Invitrogen™)

Coelenterazine h is one of several synthetic coelenterazine analogs that confer different Ca2+ affinities and spectral properties on the aequorin complex. Like native coelenterazine, these five novel derivatives can be used to reconstitute the aequorin complex both in vivo and in vitro.

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.

Indo-1, Pentapotassium Salt, cell impermeant (Invitrogen™)

This cell-impermeant indo-1 pentapotassium salt 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+. The dye is also available in the cell-permeant AM ester form—in our special packaging (I1223), in 1 mg amounts (I1203) and in 1 mM solution (I1226).

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.