This section describes a variety of probes for Ca2+, Na+, K+ and Cl ion channels and carriers. Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19, pH Indicators—Chapter 20 and Indicators for Na+, K+, Cl– and Miscellaneous Ions—Chapter 21 contain our extensive selection of indicators for these physiologically important ions, providing a means of correlating ion channel activation with subsequent changes in intracellular ion concentration. Ion flux also affects the cell's membrane potential, which can be measured with the probes described in Probes for Membrane Potential—Chapter 22.

Probes for Ca2+ Channels and Carriers

In both excitable and nonexcitable cells, intracellular Ca2+ levels modulate a multitude of vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility and cell shape and volume regulation—and thereby play a key role in regulating cell responses to external activating agents. These dynamic changes in intracellular Ca2+ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane, as well as by mobilization of Ca2+ from intracellular stores. One of the best-studied examples of Ca2+-dependent signal transduction is the depolarization of excitable cells, such as those of neuronal, cardiac, skeletal and smooth muscle tissue, which is mediated by inward Ca2+ and Na+ currents. The Ca2+ current is attributed to the movement of ions through N-, L-, P- and T-type Ca2+ channels, which are defined both pharmacologically and by their biophysical properties, including conductance and voltage sensitivity. Here we describe several fluorescent ligands for imaging the spatial distribution and localization of Ca2+ channels in cells, as well as Premo Cameleon Calcium Sensor, a genetically encoded, protein-based ratiometric sensor for calcium measurements. Our complete selection of Ca2+ indicators is described in Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19.


Fluorescent Dihydropyridine for L-Type Ca2+ Channels

The L-type Ca2+ channel is readily blocked by the binding of dihydropyridine to the channel's pore-forming α1-subunit. To facilitate the study of channel number and distribution in single cells, we offer fluorescent dihydropyridine derivatives. The high-affinity (–)-enantiomer of dihydropyridine is available labeled with either the green-fluorescent DM-BODIPY (D7443, structure) or the orange-fluorescent ST-BODIPY (S7445) fluorophore. Knaus and colleagues have shown that these BODIPY dihydropyridines bind to L-type Ca2+ channels with high affinity and inhibit the Ca2+ influx in GH3 cells.ref For neuronal L-type Ca2+ channels, the (–)-enantiomers of the DM-BODIPY dihydropyridine and ST-BODIPY derivatives each exhibit a Ki of 0.9 nM. Their affinities for skeletal muscle L-type Ca2+ channels are somewhat lower. Although DM-BODIPY dihydropyridine exhibits a more intense fluorescence, the particularly high degree of stereoselectivity retained by the ST-BODIPY derivatives has proven useful for the in vivo visualization of L-type Ca2+ channels.ref DM-BODIPY dihydropyridine has proven effective as a substrate for functional analysis of ABC drug transporters.ref


BODIPY FL Verapamil

Like dihydropyridine, phenylalkylamines also bind to the α1-subunit of L-type Ca2+ channels and block Ca2+ transport. We offer a green-fluorescent BODIPY FL derivative (B7431, structure) of verapamil, a phenylalkylamine known to inhibit P-glycoprotein–mediated drug efflux.

The 170,000-dalton P-glycoprotein is typically overexpressed in tumor cells that have acquired resistance to a variety of anticancer drugs (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). P-glycoprotein is thought to mediate the ATP-dependent efflux or sequestration of structurally unrelated molecules, including actinomycin D, anthracyclines, colchicine, epipodophyllotoxins and vinblastine. Verapamil appears to inhibit drug efflux by acting as a substrate of P-glycoprotein, thereby overwhelming the transporter's capacity to expel the drugs. BODIPY FL verapamil also appears to serve as a substrate for P-glycoprotein. This fluorescent verapamil derivative preferentially accumulates in the lysosomes of normal, drug-sensitive NIH 3T3 cells but is rapidly transported out of multidrug-resistant cells.ref


Eosin Derivatives: Inhibitors of the Calcium Pump

Eosin isothiocyanate (E18) is a potent reversible inhibitor of the erythrocyte plasma membrane calcium pump, with a half-maximal inhibitory concentration of <0.2 µM.ref Eosin isothiocyanate also reacts irreversibly at the ATP-binding site of this calcium pump. The succinimidyl ester of carboxyeosin diacetate (C22803), a cell membrane–permeant eosin derivative, also inhibits the erythrocyte plasma membrane Ca2+ pump.ref Fluorescein isothiocyanate (F143, BODIPY Dye Series—Section 1.4) is a weaker inhibitor of the erythrocyte plasma membrane calcium pump.


Premo Cameleon Calcium Sensor

The Premo product line combines genetically encoded ion indicators and environmental sensors with efficient BacMam delivery (BacMam Gene Delivery and Expression Technology—Note 11.1) for intracellular measurements in mammalian cells. Premo Cameleon Calcium Sensor (P36207, P36208) is a ratiometric calcium-sensitive fluorescent protein that is delivered by BacMam baculovirus-mediated transduction to a variety of mammalian cell types. This content and delivery system provides an effective and robust technique for measuring Ca2+ mobilization in transduced cells using microplate assays or fluorescence microscopy (photo).

The Premo Cameleon Calcium Sensor is based on the YC3.60 version of the fluorescent protein (FP)–based sensor (cameleon) family developed by Tsien, Miyawaki and coworkers, which is reported to have a Ca2+ dissociation constant of 240 nM.ref The sensor comprises two fluorescent proteins (enhanced cyan-fluorescent protein or ECFP and Venus variant of yellow-fluorescent protein or YFP), linked by the calmodulin-binding peptide M13 and calmodulin. Upon binding four calcium ions, calmodulin undergoes a conformational change by wrapping itself around the M13 peptide, which changes the efficiency of the fluorescence resonance energy transfer (FRET) between the CFP donor and the YFP acceptor fluorophores (Figure 16.3.1). Following this conformational change, there is an increase in YFP emission (525–560 nm) and a simultaneous decrease in CFP emission (460–500 nm) (Figure 16.3.2), making Cameleon an effective reporter of calcium mobilization. The ratiometric readout of Premo Cameleon Calcium Sensor—an increase in YFP emission (535 nm, green-yellow emission) and a decrease in CFP emission (485 nm, blue emission)—reduces assay variations due to compound or cellular autofluorescence, nonuniform cell plating, differences in expression levels between cells, instability of instrument illumination and changes in illumination pathlength.

The Premo Cameleon Calcium Sensor is designed to readily and accurately detect intracellular calcium flux from different receptors. Standard pharmacological assays for multiple GPCR agonists and antagonists have been tested. An example of the robustness and reproducibility and accuracy of the system is demonstrated using the endogenous histamine receptor in conjunction with histamine, pyrilamine, and thioperamide in HeLa cells (Figure 16.3.3). Expression levels will be maintained for several days, enabling iterative assays to be run; for instance, when examining agonist/antagonist relationships on the same cells.


Premo Cameleon Calcium Sensor Mechanism

Figure 16.3.1 Schematic of the Premo Cameleon Calcium Sensor (P36207, P36208) mechanism.

 

Fluorescence Emission Spectra of Premo 
Figure 16.3.2 Fluorescence emission spectra of Premo Cameleon Calcium Sensor (P36207, P36208). The dashed line indicates the spectra in the absence of Ca2+; the solid line shows the fluorescenc resonance energy transfer (FRET)–based change upon Ca2+ binding.



Premo Cameleon Calcium Sensor

Figure 16.3.3 Agonist and antagonist dose response curves. HeLa cells were plated in a 96-well plate at a density of 15,000 cells/well, transduced with Premo Cameleon Calcium Sensor (P36207, P36208), and incubated overnight at 37°C. The following day, a histamine dose response was performed (A). A separate plate was used to evaluate an antagonist dose response with pyrilamine (closed squares) and thioperamide (closed triangles) in the presence of an EC80 concentration of histamine (B). Pyrilamine is a known H1 receptor antagonist that couples through Gq proteins and the second messenger Ca2+. Thioperamide is a known H3 receptor antagonist that couples through Gi proteins and the second messenger cAMP.

Probes for Na+ Channels and Carriers

Amiloride Analogs: Probes for the Na+ Channel and the Na+/H+ Antiporter

Amiloride is a compound known to inhibit the Na+/H+ antiporter of vertebrate cells by acting competitively at the Na+-binding site.ref The antiporter extrudes protons from cells using the inward Na+ gradient as a driving force, resulting in intracellular alkalinization. In 1967, Cragoe and co-workers reported the synthesis of amiloride and several amiloride analogs, which are pyrazine diuretics that inhibit the Na+ channel in urinary epithelia.ref Since then, more than 1000 different amiloride analogs have been synthesized and many of these tested for their specificity and potency in inhibiting the Na+ channel, Na+/H+ antiporter and Na+/Ca2+ exchanger.ref Unmodified amiloride inhibits the Na+ channel with an IC50 of less than 1 µM. Additionally, amiloride is an important tool for studying the Na+/H+ antiporter. Structure–activity relationships have demonstrated that amiloride analogs with hydrophobic groups in the drug are the most potent and specific inhibitors for the Na+/H+ antiporter.ref For example, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, E3111, structure) is 200-fold more potent than amiloride for inhibiting this antiporter.


Ouabain Probes for Na+/K+-ATPase

Ouabain is a member of a class of glycosylated steroids collectively known as cardiac glycosides due to their therapeutic efficacy in the treatment of congestive heart failure. Ouabain achieves this effect by binding to the catalytic α-subunit of Na+/K+-ATPase and inhibiting its transport of Na+ across the plasma membrane. 9-Anthroyl ouabain (A1322) is useful for localizing Na+/K+-ATPase and for studying its membrane orientation, mobility and dynamics.ref Anthroyl ouabain has also been employed to investigate Na+/K+-ATPase's active site, inhibition and conformational changes,ref as well as to investigate the kinetics of cardiac glycoside binding.ref BODIPY FL ouabain (B23461, structure) has been used in combination with Alexa Fluor 555 cholera toxin B (C22843, Probes for Following Receptor Binding and Phagocytosis—Section 16.1) for visualizing Na+/K+-ATPase and ganglioside GM1 domain localization in lymphocyte plasma membranes.ref


Using BacMam Technology to Deliver and Express Sodium Channel cDNA

Sodium channel cDNAs that have been engineered into a baculovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1) are also available, including the Nav1.2 cDNA (B10341) and the Nav1.5 cDNA (B10335).

The BacMam system uses a modified insect cell baculovirus as a vehicle to efficiently deliver and express genes in mammalian cells with minimum effort and toxicity. The use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used extensively in a drug discovery setting.ref Furthermore, constitutively expressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal drift and other inconsistencies that hamper successful experimentation and screening. Thus, transient expression systems such as the BacMam gene delivery and expression system are increasingly methods of choice to decrease variability of expression in such assays.

U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly efficient expression of BacMam delivered targets in a null background ideal for screening in a heterologous expression system. The U2OS cell line is recommended for use if your particular cell line does not efficiently express the BacMam targets. Examples of other cell lines that are efficiently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.

Probes for K+ Channels and Carriers

Glibenclamide Probes for the ATP-Dependent K+ Channel

Glibenclamide blocks the ATP-dependent K+ channel, thereby eliciting insulin secretion.ref We have prepared the green-fluorescent BODIPY FL glibenclamide (BODIPY FL glyburide, E34251; structure) and red-fluorescent BODIPY TR glibenclamide (BODIPY TR glyburide, E34250) as probes for the ATP-dependent K+ channel. BODIPY TR glibenclamide has been used to detect sulfonylurea receptors associated with ATP-dependent K+ channels in bovine monocytes and in β-cells ref and to label a novel mitochondrial ATP-sensitive potassium channel in brain.ref

The sulfonylurea receptors of ATP-dependent K+ channels are prominent on the endoplasmic reticulum (ER). Therefore, because these probes are also effective live-cell stains for ER, BODIPY FL glibenclamide and BODIPY TR glibenclamide are also referred to as ER-Tracker Green and ER-Tracker Red, respectively; see Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4 for a description of this application. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling with these probes.


FluxOR Potassium Ion Channel Assay

The FluxOR Potassium Ion Channel Assay Kits (F10016, F10017) provide a fluorescence-based assay for high-throughput screening of potassium ion channel and transporter activities.ref The FluxOR Potassium Ion Channel Assay Kits take advantage of the well-described permeability of potassium channels to thallium (Tl+) ions. When thallium is present in the extracellular solution containing a stimulus to open potassium channels, channel activity is detected with a cell-permeant thallium indicator dye that reports large increases in fluorescence emission at 525 nm as thallium flows down its concentration gradient and into the cells (Figure 16.3.4). In this way, the fluorescence reported in the FluxOR system becomes a surrogate indicator of activity for any ion channel or transporter that is permeable to thallium, including the human ether-a-go-go–related (hERG) channel, one of the human cardiac potassium channels. The FluxOR potassium ion channel assay has been validated for homogeneous high-throughput profiling of hERG channel inhibition using BacMam-mediated transient expression of hERG ref (see below). The FluxOR Potassium Ion Channel Assay Kits can also be used to study potassium co-transport processes that accommodate the transport of thallium into cells.ref Furthermore, resting potassium channels and inward rectifier potassium channels like Kir2.1 can be assayed by adding stimulus buffer with thallium alone, without any depolarization to measure the signal.

The FluxOR reagent, a thallium indicator dye, is loaded into cells as a membrane-permeable AM ester. Loading is assisted by the proprietary PowerLoad concentrate, an optimized formulation of nonionic Pluronic surfactant polyols that act to disperse and stabilize AM ester dyes for optimal loading in aqueous solution. This PowerLoad concentrate is also available separately (P10020) to aid the solubilization of water-insoluble dyes and other materials in physiological media.

Once inside the cell, the nonfluorescent AM ester of the FluxOR dye is cleaved by endogenous esterases into a weakly fluorescent (basal fluorescence), thallium-sensitive indicator. The thallium-sensitive form is retained in the cytosol, and its extrusion is inhibited by water-soluble probenecid (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), which blocks organic anion pumps. For most applications, cells are loaded with the dye at room temperature. For best results, the dye-loading buffer is then replaced with fresh, dye-free assay buffer (composed of physiological HBSS containing probenecid), and cells are ready for the high-throughput screening assay.

Each FluxOR Potassium Ion Channel Assay Kit contains:

  • FluxOR reagent
  • FluxOR assay buffer
  • PowerLoad concentrate
  • Probenecid
  • FluxOR chloride-free buffer
  • Potassium sulfate (K2SO4) concentrate
  • Thallium sulfate (Tl2SO4) concentrate
  • Dimethylsulfoxide (DMSO)
  • Detailed protocols (FluxOR Potassium Ion Channel Assay)

The FluxOR Kits provide a concentrated thallium solution along with sufficient dye and buffers to perform ~4000 (F10016) or ~40,000 (F10017) assays in a 384-well microplate format. These kits allow maximum target flexibility and ease of operation in a homogeneous format. The FluxOR potassium ion channel assay has been demonstrated for use with CHO and HEK 293 cells stably expressing hERG, as well as U2OS cells transiently transduced with BacMam hERG reagent ref (B10019, B10033; see below) (Figure 16.3.5).


Thallium Redistribution in the FluxOR Assay

Figure 16.3.4 Thallium redistribution in the FluxOR assay. Basal fluorescence from cells loaded with FluxOR reagent (provided in the FluxOR Potassium Ion Channel Assay Kits; F10016, F10017) is low when potassium channels remain unstimulated, as shown in the left panel. When thallium is added to the assay with the stimulus, the thallium flows down its concentration gradient into the cells, activating the dye as shown in the right panel.


FluxOR™ potassium ion channgel assays

Figure 16.3.5 FluxOR potassium ion channel assays (F10016, F10017) performed on fresh and frozen U2OS cells transduced with the BacMam hERG reagent (B10019, B10033). A) Raw data (RFU = relative fluorescence units) obtained in the FluxOR assay determination of thallium flux in U2OS cells transduced with BacMam-hERG and kept frozen until the day of use. The arrow indicates the addition of the thallium/potassium stimulus, and upper and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves. B) Raw pre-stimulus peak and baseline values were boxcar averaged and normalized, and indicate the fold increase in fluorescence over time. C) Data generated in a dose-response determination of cisapride block on BacMam hERG expressed in U2OS cells freshly prepared from overnight expression after viral transduction. D) Parallel data obtained from cells transduced with BacMam-hERG, stored for 2 weeks in liquid nitrogen, thawed, and plated 4 hours prior to running the assay. Error bars indicate standard deviation, n = 4 per determination.

 

Using BacMam Technology to Deliver and Express Potassium Channel cDNA

Potassium channel cDNAs that have been engineered into a baculovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1) are also available for use with the FluxOR Potassium Ion Channel Assay Kits, including the human ether-a-go-go related gene (hERG) (Figure 16.3.6), several members of the voltage-gated K+ channel (Kv) gene family and two members of the inwardly rectifying K+ channel (Kir) gene family:

  • BacMam hERG ref (for 10 microplates, B10019; for 100 microplates, B10033)
  • BacMam Kv1.1 (for 10 microplates, B10331)
  • BacMam Kv1.3 (for 10 microplates, B10332)
  • BacMam Kv2.1 (for 10 microplates, B10333)
  • BacMam Kv7.2 and Kv7.3 (for 10 microplates, B10147)
  • BacMam Kir1.1 (for 10 microplates, B10334)
  • BacMam Kir2.1 (for 10 microplates, B10146)

The BacMam system uses a modified insect cell baculovirus as a vehicle to efficiently deliver and express genes in mammalian cells with minimum effort and toxicity. The use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used extensively in a drug discovery setting.ref Furthermore, constitutively expressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal drift and other inconsistencies that hamper successful experimentation and screening. Thus, inducible, division-arrested or transient expression systems such as BacMam technology are increasingly methods of choice to decrease variability of expression in such assays.

U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly efficient expression of BacMam delivered targets in a null background ideal for screening in a heterologous expression system. The U2OS cell line is recommended for use if your particular cell line does not efficiently express the BacMam targets. Examples of other cell lines that are efficiently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.


BacMam-hERG gene delivery and expression

Figure 16.3.6 BacMam-hERG gene delivery and expression. This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell and expression of the hERG gene (B10019, B10033). The hERG gene resides within the baculoviral DNA, downstream of a CMV promoter that drives its expression when introduced into a mammalian target cell. BacMam viral particles are taken up by endocytic pathways into the cell, and the DNA within them is released for transcription and expression. The translated protein is then folded for insertion into the membrane, forming functional hERG ion channels. This process begins within 4–6 hours and in many cell types is completed after an overnight period.

Probes for Anion Transporters

Stilbene Disulfonates: Anion-Transport Inhibitors

We offer three stilbene disulfonates that have been employed to inhibit (frequently irreversibly) anion transport ref in a large number of mammalian cell types:

Our stilbene disulfonate probes, which are 95–99% pure by HPLC, have significantly higher purity and more defined composition than those available from other commercial sources. DNDS was among the inhibitors used to characterize three different anion exchangers in the membranes of renal brush border cells and to compare these exchangers with the band-3 anion-transport protein of erythrocyte membranes.ref

These stilbene disulfonates can, in some cases, bind specifically to proteins that are not anion transporters. For example, DIDS and H2DIDS complex specifically with the CD4 glycoprotein on T-helper lymphocytes and macrophages, blocking HIV type-1 growth at multiple stages of the virus life cycle.ref

 

DiBAC4(5)

The membrane potential–sensing dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5), B436; structure) initially inhibits Cl exchange with an IC50 of 0.146 µM. However, this inhibition increases with time to an IC50 of 1.05 nM, making DiBAC4(5) a more potent inhibitor than DIDS, which has an IC50 of 31 nM under similar conditions.ref

 

Eosin Maleimide

Although usually selectively reactive with thiols, eosin-5-maleimide (E118, Thiol-Reactive Probes Excited with Visible Light—Section 2.2) is known to react with a specific lysine residue of the band-3 protein in human erythrocytes, inhibiting anion exchange in these cells and providing a convenient tag for observing band-3 behavior in the membrane.ref Eosin-5-isothiocyanate (E18) has similar reactivity with band-3 proteins.ref

 

Premo Halide Sensor

The fluorescent protein–based Premo Halide Sensor (P10229) is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels and their modulators in cells. Chloride channels are involved in cellular processes as critical and diverse as transepithelial ion transport, electrical excitability, cell volume regulation and ion homeostasis. Given their physiological significance, it follows that defects in their activity can have severe implications, including such conditions as cystic fibrosis and neuronal degeneration. Thus, chloride channels represent important targets for drug discovery.ref Other methods for detecting chloride are described in Detecting Chloride, Phosphate, Nitrite, and Other Anions—Section 21.2.

Premo Halide Sensor combines a yellow-fluorescent protein (YFP) variant sensitive to halide ions with the efficient and noncytopathic BacMam delivery and expression technology (BacMam Gene Delivery and Expression Technology—Note 11.1). Premo Halide Sensor is based on the Venus variant of Aequorea Victoria green-fluorescent protein (GFP), which displays enhanced fluorescence, increased folding, and reduced maturation time when compared with YFP.ref Additional mutations H148Q and I152L were made within the Venus sequence to increase the sensitivity of the Venus fluorescent protein to changes in local halide concentration, in particular iodide ions.ref Because chloride channels are also permeable to the iodide ion (I), iodide can be used as a surrogate of chloride. Upon stimulation, a chloride channel or transporter opens and iodide flows down the concentration gradient into the cells, where it quenches the fluorescence of the expressed Premo Halide Sensor protein (Figure 16.3.7). The decrease in Premo Halide Sensor fluorescence is directly proportional to the ion flux, and therefore the chloride channel or transporter activity. Premo Halide Sensor shows a similar excitation and emission profile to YFP (Figure 16.3.8) and can be detected using standard GFP/FITC or YFP filter sets. Halide-sensitive YFP-based constructs in conjunction with iodide quenching have been used in high-throughput screening (HTS) to identify modulators of calcium-activated chloride channels.ref

Premo Halide Sensor (P10229) is pre-packaged and ready for immediate use. It contains all components required for cellular delivery and expression, including baculovirus carrying the genetically encoded biosensor, BacMam enhancer and stimulus buffer. Premo Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells (HBEC), providing the flexibility to assay chloride permeable channels in a wide range of cellular models. To uncouple cell maintenance and preparation from cell screening, BacMam-transduced cells can be divided into aliquots and frozen for later assay. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready” and can be subsequently plated as little as four hours prior to screening. Screening can be conducted in complete medium and without any wash steps. Chloride channel assays with Premo Halide Sensor are compatible with standard fluorescence HTS platforms.


Principle of Premo Halide Sensor Sensor

Figure 16.3.7 Principle of Premo Halide Sensor Sensor (P10229): Iodide redistribution upon chloride channel activation. Basal fluorescence from Premo Halide Sensor is high when chloride channels are closed or blocked. Upon activation (opening) of chloride channels, the iodide ions enter the cell, down its concentration gradient, and quench the fluorescence from Premo Halide Sensor.




Quenching of Premo Halide Sensor Fluorescence

Figure 16.3.8 Quenching of Premo Halide Sensor fluorescence by increasing concentrations of iodide and chloride. U2OS cells were transduced with Premo Halide Sensor. After 24 hours, cells were trypsinized and lysed by resuspension in sterile distilled water. Fluorescence quenching of the lysate was examined using increasing concentrations of NaCl (A) and NaI (B). Iodide induces substantially greater quenching of Premo Halide Sensor fluorescence than chloride.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A1322icon788.89F,D,LDMSO3627500471MeOH 
B436icon542.67LDMSO, EtOH590160,000616MeOH1
B7431icon icon769.18F,D,LDMSO, EtOH50474,000511MeOH 
B23461icon858.74F,D,LDMSO50380,000510MeOH 
C22803icon873.05F,DDMSO<300 none  
D337icon498.47F,DDH2O34161,000415H2O2
D338icon500.48F,DDH2O28641,000noneMeOH2
D673icon474.32LH2O35232,000noneH2O 
D7443icon icon686.48F,D,L,ADMSO, EtOH50483,000511MeOH 
E18icon icon704.97F,DD,LpH >6, DMF52195,000544pH 92
E3111icon336.22D,LH2O, MeOH37823,000423MeOH 
E34250icon915.23F,D,LDMSO, H2O58760,000615MeOH 
E34251icon783.10F,D,LDMSO, H2O50476,000511MeOH 
S7445icon760.57F,D,L,ADMSO, EtOH565143,000570MeOH 
  1. Oxonols may require addition of a base to be soluble.
  2. Isothiocyanates are unstable in water and should not be stored in aqueous solution.

Ordering Information

Sku Name Size Price Qty
B7431 BODIPY® FL Verapamil, Hydrochloride 1 mg CNY 2,965.00
B23461 BODIPY® FL Ouabain 100 µg CNY 3,195.00
C22803 CEDA, SE (5-(and-6)-Carboxyeosin Diacetate, Succinimidyl Ester), mixed isomers 5 mg CNY 3,320.00
D337 DIDS (4,4'-Diisothiocyanatostilbene-2,2'-Disulfonic Acid, Disodium Salt) 100 mg CNY 646.00
D338 H2DIDS (H2DIDS (4,4'-Diisothiocyanatodihydrostilbene-2,2'-Disulfonic Acid, Disodium Salt) 100 mg CNY 3,175.00
D673 DNDS (4,4'-Dinitrostilbene-2,2'-Disulfonic Acid, Disodium Salt) 1 g CNY 1,388.00
D7443 DM-BODIPY® (-)-Dihydropyridine, high affinity enantiomer 25 µg CNY 3,395.00
E18 Eosin-5-Isothiocyanate 100 mg CNY 3,235.00
F10016 FluxOR™ Potassium Ion Channel Assay 10 microplates CNY 5,200.00
F10017 FluxOR™ Potassium Ion Channel Assay 100 microplates Contact Us  
P10229 Premo™ Halide Sensor 1 kit CNY 6,830.00
E34250 ER-Tracker™ Red (BODIPY® TR Glibenclamide), for live-cell imaging 100 µg CNY 3,840.00
E34251 ER-Tracker™ Green (BODIPY® FL Glibenclamide), for live-cell imaging 100 µg CNY 3,840.00
B10019 BacMam-hERG Potassium Channel Kit 1 kit CNY 8,580.00
B10332 BacMam Kv1.3 Ion Channel Reagents 1 kit CNY 7,200.00
B10333 BacMam Kv2.1 Ion Channel Reagents 1 kit CNY 7,200.00
B10334 BacMam Kir1.1 Ion Channel Reagents 1 kit CNY 7,200.00
P10020 PowerLoad™ Concentrate, 100X 5 mL CNY 1,622.00
For Research Use Only. Not for use in diagnostic procedures.