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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".

Dihydrorhodamine 123 (Invitrogen™)

Dihydrorhodamine 123 is an uncharged and nonfluorescent reactive oxygen species (ROS) indicator that can passively diffuse across membranes where it is oxidized to cationic rhodamine 123 which localizes in the mitochondria and exhibits green fluorescence.

DAF-FM (4-Amino-5-Methylamino-2',7'-Difluorofluorescein) (Invitrogen™)

DAF-FM is a reagent that is used to detect and quantify low concentrations of nitric oxide (NO). It is essentially nonfluorescent until it reacts with NO to form a fluorescent benzotriazole. DAF-FM fluorescence can be detected by any instrument that can detect fluorescein, including flow cytometers, microscopes, fluorescent microplate readers and fluorometers.

DAF-FM Specifications:
• Ex/Em of DAF-FM: ~495/515 nm
• Lyophilized product should be dissolved using DMSO and then added to an aqueous buffer to create a working solution
• DAF-FM can be introduced into cells by pressure injection or perfusion from a patch-clamp pipette (choose DAF-FM diacetate if you need a cell-permeant version)
• Buffers containing bovine serum albumin (BSA) and phenol red may affect the fluorescence and should be used with caution
• The fluorescence quantum yield of DAF-FM is ~0.005, but increases about 160-fold, to ~0.81, after reacting with NO


Applications of DAF-FM:
• Assessment of NO production in transaldolase-deficient lymphoblasts by flow cytometry
• Detection of NO accumulation in embryonic cortical neurons following neurotrophin stimulation
in vivo imaging of NO in zebrafish
• Intravital microscopic detection of NO generation associated with angiogenesis in mice
• Quantitation of ATP-induced NO release in rabbit platelets

Important Advantages of DAF-FM Over DAF-2
The spectra of the NO adduct of DAF-FM are independent of pH above pH 5.5. Also, the NO adduct of DAF-FM is significantly more photostable than that of DAF-2, which means additional time for image capture. Finally, DAF-FM is a more sensitive reagent for NO than is DAF-2 (NO detection limit for DAF-FM ~3 nM versus ~5 nM for DAF-2).

Probes for Nitric Oxide Research
We offer several products for the detection of reactive oxygen species (ROS) and nitric oxide (NO). Review these products in Probes for Nitric Oxide Research—Section 18.3 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).

DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate) (Invitrogen™)

DAF-FM is a reagent that is used to detect and quantify low concentrations of nitric oxide (NO). It is essentially nonfluorescent until it reacts with NO to form a fluorescent benzotriazole. DAF-FM fluorescence can be detected by any instrument that can detect fluorescein, including flow cytometers, microscopes, fluorescent microplate readers and fluorometers.

DAF-FM Specifications:
• Ex/Em of DAF-FM: ~495/515 nm
• Lyophilized product should be dissolved using DMSO and then added to an aqueous buffer to create a working solution
• DAF-FM diacetate is cell permeant and passively diffuses across cellular membranes; once inside the cell, it is converted to a cell-impermeant form
• Buffers containing bovine serum albumin (BSA) and phenol red may affect the fluorescence and should be used with caution
• The fluorescence quantum yield of DAF-FM is ~0.005, but increases about 160-fold, to ~0.81, after reacting with NO


Applications of DAF-FM:
• Assessment of NO production in transaldolase-deficient lymphoblasts by flow cytometry
• Detection of NO accumulation in embryonic cortical neurons following neurotrophin stimulation
in vivo imaging of NO in zebrafish
• Intravital microscopic detection of NO generation associated with angiogenesis in mice
• Quantitation of ATP-induced NO release in rabbit platelets

Important Advantages of DAF-FM Over DAF-2
The spectra of the NO adduct of DAF-FM are independent of pH above pH 5.5. Also, the NO adduct of DAF-FM is significantly more photostable than that of DAF-2, which means additional time for image capture. Finally, DAF-FM is a more sensitive reagent for NO than is DAF-2 (NO detection limit for DAF-FM ~3 nM versus ~5 nM for DAF-2).

Probes for Nitric Oxide Research
We offer several products for the detection of reactive oxygen species (ROS) and nitric oxide (NO). Review these products in Probes for Nitric Oxide Research—Section 18.3 in the Molecular Probes® Handbook.

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

Coelenterazine (Invitrogen™)

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

5-(and-6)-Carboxy SNARF™-1, Acetoxymethyl Ester, Acetate (Invitrogen™)

The cell-permeant pH indicator, carboxy SNARF®-1, acetoxymethyl ester, acetate has a pKa of ~7.5 after de-esterification, thus is useful for measuring pH changes between pH 7 and pH 8. Carboxy-SNARF®-1 exhibits a significant pH-dependent emission shift from yellow-orange to deep red fluorescence under acidic and basic conditions, respectively. This pH dependence allows the ratio of the fluorescence intensities from the dye at two emission wavelengths - typically 580 nm and 640 nm - to be used for quantitative determinations of pH.

Sodium Green™ Tetraacetate, cell permeant - Special Packaging (Invitrogen™)

This cell-permeant form of Sodium Green™ tetraacetate is a visible light-excitable Na+ indicator.

PBFI, Tetraammonium Salt, cell impermeant (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.

Fluorescent Ion Indicators Specifications:
• Label (Ex/Em): PBFI (~340,380/500 nm)
• Lyophilized product may be dissolved in distilled water or aqueous buffer for use
• Product is typically loaded into cells by diffusion from a patch pipette for correlated fluorescence imaging and electrophysiological recording


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.

Calcium Green™-5N, Hexapotassium Salt, cell impermeant (Invitrogen™)

The cell-impermeant visible light-excitable Ca2+ indicator, Calcium Green™-5N, exhibits an increase in fluorescence emission intensity upon binding Ca2+ with little shift in wavelength. Calcium Green™-5N is essentially nonfluorescent in the absence of Ca2+ and the low-affinity indicator has dissociation constant for Ca2+ in the absence of Mg2+ of ~14 µM.

CellROX™ Reagent Variety Pack, for oxidative stress detection (Invitrogen™)

The CellROX® Variety Pack contains one vial (50 µL) each of CellROX® Green Reagent, CellROX® Orange Reagent, and CellROX® Deep Red Reagent. These reagents are novel fluorogenic probes for measuring cellular oxidative stress in live cells. These cell-permeant dyes exhibit bright fluorescence upon oxidation by reactive oxygen species (ROS). (See table below for details of each reagent.)

The CellROX® reagents are:

• Optimized for detection of oxidative stress in live cells and may be added directly to serum-containing growth media
• Provided as a stable, ready-to-use DMSO solutions with simple protocols, compatible with standard workflows in fluorescence microscopy
• Compatible with a variety of detection platforms, including traditional fluorescence microscopy, high content screening (HCS), and flow cytometry

Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases, including inflammation, atherosclerosis, aging, and age-related degenerative disorders.

 Catalog # Absorption/Emission (Color) Live cell compatible? Formaldehyde fixable?Detergent resistant? Platforms
CellROX® Deep Red ReagentC10422 644/665 nm (Deep Red) Yes YesNo Imaging, HCS, HTS, flow cytometry, Attune™
CellROX® Orange ReagentC10443 545/565 nm (Orange) Yes NoNo Imaging, HCS, flow cytometry, Tali™
CellROX® Green ReagentC10444 485/520 nm (Green) Yes YesYes Imaging, HCS, HTS, FLOW, Tali™, FLoid™, Attune™


For Research Use Only. Not intended for any animal or human 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).

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.

Magnesium Green™, AM, cell permeant - Special Packaging (Invitrogen™)

The cell-permeant Magnesium Green™, AM exhibits a higher affinity for Mg2+ (Kd ~1.0 mM) than mag-fura-2 (Kd ~1.9 mM) or mag-indo-1 (Kd ~2.7 mM). Upon binding Mg2+, Magnesium Green exhibits and increase in fluorescence emission intensity without a shift in wavelength with absorption/emission maxima ~506/531 nm.

Fc OxyBURST™ Green Assay Reagent (Invitrogen™)

The Fc OxyBURST® Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydrofluorescein (H2DCF) and then complexed with a purified rabbit polyclonal anti-BSA antibody. When this immune complex binds to Fc receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuoloe and subsequently oxidized to green-fluorescent 2''7'-dichlorofluorescein.