Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8

Nitrophenyl EGTA: A Superior Caged Calcium Reagent

As an alternative to solely monitoring Ca²⁺ changes using fluorescent indicators, scientists may want to rapidly raise or lower the intracellular Ca²⁺ concentration and study the physiological response that results. Ellis-Davies and Kaplan have developed a photolabile chelator, o-nitrophenyl EGTA (NP-EGTA, N6802) that exhibits a high selectivity for Ca²⁺, a dramatic 12,500-fold decrease in affinity for Ca²⁺ upon UV illumination (its K_d increases from 80 nM to >1 mM) and a high photochemical quantum yield (~0.2). Photolysis of NP-EGTA is slightly faster than that of DMNP-EDTA, another "caged Ca²⁺" reagent that is frequently called DM-Nitrophen (see below). Furthermore, with a K_d for Mg²⁺ of 9 mM, NP-caged EGTA does not bind physiological levels of Mg²⁺ and thus reduces interference from this abundant cation. Skinned muscle fibers equilibrated with NP-EGTA were shown to contract maximally upon irradiation with a single flash from a frequency-doubled ruby laser (347 nm illumination). Some applications of NP-EGTA include:

• Assessing Ca²⁺ binding to troponin C in skeletal muscle fibers
• Generating Ca²⁺ pulses in astrocytes that are transmitted to hippocampal neurons via NMDA receptor transduction
• Investigating the coupling of rapid Ca²⁺ release to depolarization in Limulus photoreceptors
• Measuring the Ca²⁺ buffering capacity of the neuronal cytosol
• Studying the mechanism of Ca²⁺-induced exocytosis in pancreatic beta-cells

We exclusively offer the tetrapotassium salt (N6802) and the acetoxymethyl (AM) ester (NP-EGTA AM, N6803) of NP-EGTA. The NP-EGTA salt can be complexed with Ca²⁺ to generate a caged Ca²⁺ reagent that will rapidly deliver Ca²⁺ upon photolysis (Figure 17.4). The cell-permeant AM ester of NP-EGTA does not bind Ca²⁺ unless its AM ester groups are removed. NP-EGTA AM can serve as a photolabile buffer in cells because, once converted to NP-EGTA by intracellular esterases, it will bind Ca²⁺ with high affinity until photolyzed with UV light.

DMNP-EDTA

The first caged Ca²⁺ reagent to be described by Kaplan and Ellis-Davies was 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA (DMNP-EDTA, D6814), which they named DM-Nitrophen (now a trademark of Calbiochem-Novabiochem Corp.). Because its structure more closely resembles that of EDTA than EGTA, we named it as a caged EDTA derivative (Figure 17.5). Upon illumination, DMNP-EDTA's dissociation constant for Ca²⁺ increases from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed with Ca²⁺ results in a pulse of free Ca²⁺. Furthermore, DMNP-EDTA has significantly higher affinity for Mg²⁺ (K_d = 2.5 µM) than does NP-EGTA (K_d = 9 mM). Because the photolysis product's K_d for Mg²⁺ is ~3 mM, DMNP-EDTA is an effective caged Mg²⁺ source, in addition to its applications for photolytic Ca²⁺ release. Photorelease of Ca²⁺ has been shown to occur in <180 µsec, with even faster photorelease of Mg²⁺. Moreover, DMNP-EDTA is also useful for photolytic release of other divalent cations such as Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺ and Cd²⁺. A paper by Neher and Zucker reviews the uses and limitations of DMNP-EDTA. Photolysis of DMNP-EDTA has been used extensively to release Ca²⁺ in a variety of systems. This caged Ca²⁺ reagent has been employed to:

• Evoke secretion in bovine chromaffin cells
• Generate rapid (~low milliseconds) Ca²⁺ transients, as detected by Calcium Orange-5N fluorescence
• Induce Ca²⁺ transients approximating the size and amplitude of ryanodine receptor–generated Ca²⁺ sparks in cardiac muscle by two-photon photolysis (Figure 19.84)
• Investigate temperature modulation of Ca²⁺-activated neurotransmitter release from squid neurons
• Measure Ca²⁺ pump kinetics in erythrocyte ghosts using the Calcium Green-2 and Calcium Green-5N indicators
• Stimulate neurotransmitter release in nerve terminals

Photoactivatable Calcium Scavenger

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 is a photoactivatable Ca²⁺ scavenger. Diazo-2 (D3034, ), which was introduced by Adams, Kao and Tsien, is a relatively weak chelator (K_d for Ca²⁺ = 2.2 µM). Following flash photolysis at ~360 nm, however, cytosolic free Ca²⁺ rapidly binds to the high-affinity photolysis product of diazo-2 (K_d = 73 nM). Photolysis of diazo-2 has been used to decrease cytosolic Ca²⁺ in less than three milliseconds in tensed frog muscle cells, rat fibroblasts and rabbit arterial smooth muscle. Trapping of Ca²⁺ following photolysis of diazo-2 results in relaxation of skinned cardiac or skeletal muscle and rapid depletion of Ca²⁺ in ventricular myocytes. Microinjecting a relatively low concentration of a visible light–excitable Ca²⁺ indicator—such as fluo-3, fluo-4 or one of our Calcium Green or Oregon Green 488 BAPTA indicators—along with a known quantity of diazo-2 permits measurement of the extent of depletion of cytosolic Ca²⁺ following photolysis.

Diazo-2 can be microinjected into larger cells or electroporated into smaller cells as its potassium salt (D3034). Diazo-2 also has been loaded into cells permeabilized by Staphylococcus aureus α-toxin.

BAPTA and BAPTA AM

The BAPTA buffers developed by Tsien are highly selective for Ca²⁺ over Mg²⁺ and can be used to control the level of both intracellular and extracellular Ca²⁺ (Ca2+ affinities of BAPTA chelators—Table 19.7, Figure 19.85). The BAPTA buffers are more selective for Ca²⁺ than EDTA and EGTA, and their metal binding is also much less pH sensitive. Furthermore, BAPTA buffers bind and release Ca²⁺ ions about 50–400 times faster than EGTA. Both BAPTA and its membrane-permeant AM ester are extensively used to clamp intracellular Ca²⁺ concentrations, providing insights on the role of free cytosolic Ca²⁺ in a number of important cell systems.

BAPTA is available as a cell-impermeant potassium, cesium or sodium salt (B1204, ; B1212; B1214); the Cs⁺ salt of BAPTA has frequently been used for patch-clamp experiments. In addition, we offer the cell-permeant BAPTA AM ester (B1205). We also make BAPTA AM available in a special packaged set of 20 vials, each containing 1 mg (B6769). In addition to these products, we offer a polystyrene conjugate of BAPTA for selective removal of Ca²⁺ from solutions, which we call Calcium Sponge S (C3047, see below).

Other BAPTA Derivatives

Other BAPTA derivatives are listed in Ca2+ affinities of BAPTA chelators—Table 19.7, along with their dissociation constants for Ca²⁺. The most powerful Ca²⁺ chelator among these is 5,5'-dimethyl BAPTA, available as its cell-permeant AM ester (D1207).

BAPTA derivatives with intermediate affinity for Ca²⁺, such as 5,5'-dibromo BAPTA (D1211), have been extensively used to study Ca²⁺ mobilization, spatial Ca²⁺ buffering and Ca²⁺ shuttling in a variety of cells, including Xenopus eggs, fucoid eggs, plants and hair cells. 5,5'-Dibromo BAPTA has also been used to block nuclear vesicle fusion in Xenopus egg extracts, to determine the affinity of a Ca²⁺-binding protein and to load Ca²⁺ into stamenal hairs of Setcreasea purpurea in a study of cytoplasmic streaming. 5,5'-Dibromo BAPTA and other lower-affinity chelators protect neurons against excitotoxic and ischemic injury, without markedly attenuating intracellular Ca²⁺ levels.

Fluorinated BAPTA derivatives, such as the AM ester of 5,5'-difluoro BAPTA (D1209), have been employed for optical imaging studies but are most widely used for NMR analysis of Ca²⁺ in live cells and tissues, including kidney, heart, brain, erythrocytes and platelets. The ¹⁹F NMR shifts of the 5,5'-difluoro BAPTA have been reported to correlate with intracellular Ca²⁺ in BALB/c thymocytes, normal and sickle erythrocytes and ferret hearts. In addition, ¹⁹F NMR has been used with 5,5'-difluoro BAPTA to detect Pb²⁺ uptake by platelets.

BAPTA Acetoxymethyl Esters

We offer three different cell-permeant acetoxymethyl (AM) esters of BAPTA:

• BAPTA AM (B1205, B6769)
• 5,5'-Dimethyl BAPTA AM (D1207)
• 5,5'-Difluoro BAPTA AM (D1209)

EGTA AM

The AM ester derivative of EGTA (E1219, ) can be passively loaded into cells to generate intracellular EGTA. The slower on-rate of EGTA relative to the BAPTA-based buffers reduces its ability to inhibit Ca²⁺ diffusion in cells. Because Ca²⁺ binding by intracellular EGTA is relatively slow it is possible to distinguish between buffering of rapid Ca²⁺ transients, which can occur with BAPTA-derived buffers, and the slower effects of general Ca²⁺ buffering. EGTA AM has been reported to:

• Block oxidative stress caused by hydroperoxide formation in kidney proximal tubular cells
• Confer neuroprotective activity
• Inhibit the substance K⁺–induced Ca²⁺ rise in glioma cells
• Selectively remove Ca²⁺ from presynaptic terminals, allowing unambiguous detection of strontium fluxes

DTPA Isothiocyanate

DTPA isothiocyanate (I24221) can be coupled to antibodies and other biomolecules by conventional amine-reactive chemistry, thereby introducing a high-affinity binding site for a variety of metal ions. Unlike reactive anhydride forms of DTPA, the isothiocyanate derivative yields conjugates that retain all five carboxylate groups (), resulting in more stable metal complexation. Antibodies labeled with the fluorescent lanthanides europium and terbium (T1247, Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7) are widely utilized in time-resolved fluorescence–based immunoassays. DTPA-gadolinium (Gd³⁺) complexes are extensively used as contrast agents for magnetic resonance imaging. DTPA isothiocyanate–labeled antibodies also have potentially important therapeutic applications for targeted delivery of radionuclides such as indium-111 and yttrium-90.

Calcium Sponge Polymer

We offer a biologically compatible conjugate of the BAPTA chelator to selectively remove specific polyvalent ions from solution, as well as from the binding sites of indicators, proteins and polynucleotides. This BAPTA polystyrene conjugate—Calcium Sponge S—is selective for Ca²⁺ and certain other ions, including Zn²⁺ and some heavy metals, in the presence of relatively high levels of Mg²⁺. Many contaminating polycations can be selectively removed from aqueous solutions simply by stirring a solution with the water-insoluble Calcium Sponge S polymer (BAPTA polystyrene, C3047). For example, free Ca²⁺ can be reduced to less than 40 nM (measured with fura-2) by passing 3 mL of a 100 µM CaCl₂ solution through one gram of Calcium Sponge S. The polymer can be regenerated several times by washing it with pH 4 buffer, then readjusting to neutral pH with base.

TPEN

TPEN (T1210, ) selectively chelates intracellular heavy metal ions such as Zn²⁺, Cu²⁺ and Fe²⁺ without disturbing Ca²⁺ and Mg²⁺ concentrations, revealing distortions in intracellular Ca²⁺ measurements caused by high-affinity binding of these ions to fluorescent indicators. TPEN has been used to show that the effects of BAPTA on mitotic progression and nuclear assembly are specifically Ca²⁺-mediated and are not attributable to binding of essential heavy metal ions. TPEN has also been used to modulate the effects of Zn²⁺ on enzymatic activity and protein conformation.

Calibration of fluorescent Ca²⁺ indicators is a prerequisite for accurate Ca²⁺ measurements. We offer a number of kits designed to facilitate this calibration using a laboratory fluorometer or quantitative imaging system. All of these kits contain buffers and detailed protocols—including methods for calculating K_d, a sample response curve and tables to help determine the exact concentration of free Ca²⁺ under conditions of varying pH, temperature and ionic strength. A discussion of methods to correct the fura-2 dissociation constants for differences in temperature and ionic strength has been published. A computer program is now available online for calculating the free Ca²⁺ concentrations in solutions that contain several chelating species, or that contain ions such as Zn²⁺ that compete with Ca²⁺ for binding to BAPTA or EGTA (MAXC Computer Program for Calculating Free Ca2+ Concentrations—Note 19.2).

Calcium Calibration Buffer Kits for High- to Moderate-Affinity Ca²⁺ Indicators

Because cells contain very low levels of free Ca²⁺, it is essential to use calibrated Ca²⁺ buffers such as EGTA to precisely calibrate the Ca²⁺ indicators in a researcher's own equipment under their own experimental conditions. When the concentrations of Ca²⁺ and EGTA are very close to each other, the only free Ca²⁺ available is the Ca²⁺ that is in equilibrium with EGTA. Thus, the concentration of free Ca²⁺ is determined by the K_d of CaEGTA at a controlled pH, temperature and ionic strength. In order to attain Ca²⁺ and EGTA concentrations sufficiently close to each other, our Calcium Calibration Buffer Kits (C3008MP, C3009, F6774) contain CaEGTA solutions that have been accurately prepared using Roger Tsien's "pH-metric" method, which makes use of the fact that ion binding by EGTA causes an acidification of the solution. These kits can be used to obtain calibration curves and Ca²⁺ dissociation constants for all of our high- to moderate-affinity Ca²⁺ indicators, including fura-2, fura-4F, fura-5F, indo-1, fluo-3, fluo-4 and the Calcium Green-1, Calcium Green-2, Calcium Orange, Calcium Crimson, Oregon Green 488 BAPTA-1, Oregon Green 488 BAPTA-2 and Fura Red indicators.

Calcium Calibration Buffer Kit #1 (C3008MP) contains:

• 10 mM K₂EGTA buffered solution ("zero" free Ca²⁺)
• 10 mM CaEGTA buffered solution (40 µM free Ca²⁺)
• Detailed protocols for calibrating Ca²⁺ indicators (Calcium Calibration Buffer Kits)

When used according to the protocol provided, each kit provides sufficient reagents for five complete calibrations using 2 mL samples and a standard fluorometer cuvette. Many more calibrations can be done by digital imaging microscopy. This kit employs a reciprocal dilution method—an equal amount of dye is added to a portion of the zero and 40 µM free Ca²⁺ solutions, and the two are then crossdiluted to give a series of solutions—which minimizes indicator concentration errors but requires more effort in making the dilutions than does Kit #2 (see below). With ratiometric indicators, this method yields a series of curves that exhibit an accurate isosbestic point (see, for example, (Figure 19.3); it is the method regularly used in our laboratories to determine Ca²⁺ affinities. Our Calcium Calibration Buffer Kit #1 was used as a source of known free Ca²⁺ concentrations in an experiment that examined the effect of Ca²⁺ binding on the fluorescence of calretinin.

In Calcium Calibration Buffer Kit #2 (C3009), 11 prediluted K₂EGTA/CaEGTA solutions containing from 0 to 10 mM CaEGTA (0 to 40 µM free Ca²⁺) are bottled separately, making the calibrations faster, especially for cases in which all the dilutions are not required. This kit is intrinsically less accurate than Kit #1 because it requires precise addition of the same amount of fluorescent indicator to each solution. Calcium Calibration Buffer Kit #2 is preferred for calibrating the response of Ca²⁺ imaging systems, in which single drops of the fura-2–containing buffer are placed on a microscope slide. Calcium Calibration Buffer Kit #2 was used to calibrate a fiber-optic Ca²⁺ sensor constructed by covalent immobilization of a Calcium Green derivative.

Calcium Calibration Buffer Kits with Magnesium

Although our Calcium Calibration Buffer Kits with Magnesium are no longer commercially available, they can easily be created from our Calcium Calibration Buffer Kits #1 and #2 by adding 1 mM Mg²⁺ to the provided buffers. This approximately physiological Mg²⁺ level may strongly affect the K_d for Ca²⁺ of the BAPTA-derived indicators. Thus, with the addition of 1 mM Mg²⁺, these buffer kits are suitable for simulating the intracellular Mg²⁺ environment during the calibration procedure and for determining the effect of this ion on the K_d of the Ca²⁺ indicator. Such effects can be significant; for example, Tsien reports that the K_d of fura-2 increases from 135 nM in the absence of Mg²⁺ to 224 nM in the presence of 1 mM Mg²⁺.

Fura-2 Calcium Imaging Calibration Kit

The Fura-2 Calcium Imaging Calibration Kit (F6774), which is designed to facilitate rapid calibration and standardization of digital imaging microscopes, contains the same 11 prediluted buffers as in our Calcium Calibration Buffer Kit #2. However, in this kit the buffers also include 50 µM fura-2, as well as 15 µm unstained polystyrene microspheres to act both as spacers that ensure uniform separation between the slide and the coverslip and as focusing aids. We also provide a twelfth buffer, identical to the 10 mM CaEGTA standard but lacking fura-2, that serves as a control for background fluorescence.

The Influx pinocytic cell-loading reagent (I14402) facilitates the loading of water-soluble materials into live cells via a rapid and simple technique based on the osmotic lysis of pinocytic vesicles. Simply mix the water-soluble probe at high concentration with the Influx reagent blended into growth medium, then incubate the cells in the medium to allow pinocytic uptake of the surrounding solution. When the cells are subsequently transferred to a slightly hypotonic medium, pinocytic vesicles within the cells release the trapped material and fill the cytosol with the probe (Figure 19.89).

The Influx pinocytic cell-loading reagent is highly effective for loading a diverse array of probes—including calcein (, ), Alexa Fluor hydrazides (), dextran conjugates of fluorophores and ion indicators (), fura-2 salts, Oregon Green 514 dye–labeled tubulin, Alexa Fluor 488 dye–labeled actin, heparin, hydroxyurea, DNA, siRNAs, antisense oligonucleotides, SYTOX Green nucleic acid stain and propidium iodide—into a variety of cell lines. We and other researchers have successfully tested the reagent and loading method with:

• Bovine pulmonary artery endothelial cells (BPAEC)
• Human epidermoid carcinoma cells (A431)
• Human T-cell leukemia cells (Jurkat)
• Murine fibroblasts (NIH 3T3 and CRE BAG 2)
• Murine monocyte-macrophages (RAW264.7 and J774A.1)
• Murine myeloma cells (P3x63AG8)
• Rat basophilic leukemia cells (RBL)
• Dorsal root ganglion cells

More than 80% of the cells remained viable, as determined by subsequent exclusion of propidium iodide.

In addition to the Influx pinocytic cell-loading reagent and cell growth medium, all that is required to perform the loading procedure is sterile deionized water and the fluorescent probe or other polar molecule of interest. Cell labeling can be accomplished in a single 30-minute loading cycle and may be enhanced by repetitive loading. Although most types of cells load quickly and easily, optimal conditions for loading must be determined for each cell type. It is also important to note that cell-to-cell variability in the degree of loading is typical () and that higher variability is generally observed when using large compounds, such as >10,000 MW dextrans and proteins.

The Influx pinocytic cell-loading reagent is packaged as a set of 10 tubes (I14402), each containing sufficient material to load 50 samples of cells grown on coverslips following the standard protocol supplied. Cells in suspension or in culture flasks may also be easily loaded; however, the number of possible cell loadings will depend on the cell suspension volume or size of culture flask used. The information provided with the Influx reagent includes general guidelines and detailed suggestions for optimizing cell loading. Use of the coverslip mini-rack or coverslip maxi-rack (C14784, C24784; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) facilitates cell loading and slide handling when using the Influx reagent.

P2X₇ receptor–expressing cells such as macrophages and thymocytes exhibit reversible pore opening that can be exploited to provide an entry pathway for intracellular loading of both cationic and anionic fluorescent dyes with molecular weights of up to 900 daltons. Pore opening is induced by treatment with 5 mM ATP for five minutes and subsequently reversed by addition of divalent cations (Ca²⁺ or Mg²⁺). Dyes that have been successfully loaded into macrophage cells by this method include:

• Ca²⁺ indicator: fura-2 (F1200, F6799; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2)
• pH indicator: HPTS (H348, Probes Useful at Near-Neutral pH—Section 20.2)
• Aqueous tracers: lucifer yellow CH (L453, L682, L1177, L12926; Polar Tracers—Section 14.3) and 6-carboxyfluorescein (C1360, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5)
• Nucleic acid stain: YO-PRO-1 (Y3603, Nucleic Acid Stains—Section 8.1)

We offer one of the most potent and widely used P2X receptor agonists, BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, B22358, Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3). BzBzATP has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling; see Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins— Section 17.3 for more information on nucleotide analogs.

A-23187 and 4-Bromo A-23187

The Ca²⁺ ionophore A-23187 (A1493, Figure 19.91) is commonly used for in situ calibrations of fluorescent Ca²⁺ indicators, to equilibrate intracellular and extracellular Ca²⁺ concentrations and to permit Mn²⁺ to enter the cell to quench intracellular dye fluorescence. Although the intrinsic fluorescence of A-23187 is too high for use with fura-2, indo-1 and quin-2, it is suitable for use with the visible light–excitable indicators, including Calcium Green, Magnesium Green, Calcium Orange, Calcium Crimson, Oregon Green 488 BAPTA, fluo-3, fluo-4, rhod-2, X-rhod-1 and Fura Red. Brominated A-23187 (4-bromo A-23187, B1494; Figure 19.91), which is essentially nonfluorescent, is the best ionophore for use with fura-2, indo-1 and other UV light–excited Ca²⁺ indicators. Like A-23187, 4-bromo A-23187 rapidly transports both Ca²⁺ and Mn²⁺ into cells. Both A-23187 and 4-bromo A-23187 can also be used to equilibrate intracellular and extracellular Mg²⁺ concentrations, making them useful for calibrating Mg²⁺ indicators (Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7). Furthermore, 4-bromo A-23187 has occasionally been used to equilibrate intracellular Zn²⁺ with controlled extracellular levels for in situ calibration of fluorescent indicators. The zero reference level for intracellular Zn²⁺ calibrations is usually set by addition of TPEN (K_d for Zn²⁺ = 2.6 × 10^-16 M) (T1210).

Ionomycin

Ionomycin (I24222, ) is an effective Ca²⁺ ionophore that is commonly used both to modify intracellular Ca²⁺ concentrations and to calibrate fluorescent Ca²⁺ indicators when studying the regulatory properties of Ca²⁺ in cellular processes. Ionomycin also transports Pb²⁺ and some other divalent cations, as well as several lanthanide series trivalent cations, at efficiencies that are greater than or equal to those for Ca²⁺.

Probenecid (P36400) 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. For example, fluo-4 AM is a Ca²⁺ indicator widely used for in-cell measurement of agonist-stimulated and antagonist-inhibited calcium signaling. The acetoxymethyl ester (AM) dye precursor is uncharged, cell permeant, and nonfluorescent. Inside the cell, nonspecific esterases cleave the AM blocking groups, generating the charged, active form of fluo-4, which fluoresces upon Ca²⁺ binding. Although the charged dye molecules leak out of cells far more slowly than the uncharged precursor molecules, the anion transporters promote the leakage of the fluorescent dye over time. The use of probenecid to block the efflux of intracellular dyes was first reported by Di Virgilio and co-workers. Wash steps or quencher dyes may be incorporated into fluorescent assays in order to minimize baseline fluorescence. However, washing introduces an extra step that is undesirable for high-throughput applications and that may also risk loss of nonadherent cells. Quencher dyes, while offering the advantage of homogeneous (one-step, mix-and-read) assays, may interact negatively with some receptor systems of interest. The use of probenecid to suppress efflux of fluorescent dyes is a favorable method for reducing baseline fluorescence.

Our water-soluble probenecid, available as a stand-alone product (P36400) or supplied with our Fluo-4 NW (no-wash) Calcium Assay Kits (F36205, F36206; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3), has the advantages of being easy to dissolve in buffer and safer to use than the commonly used free acid form, which requires caustic 1 M NaOH to dissolve it. It is also slightly more effective than the free acid form at equimolar concentrations, probably due to its better solubility.

Pluronic F-127

Because acetoxymethyl (AM) esters have low aqueous solubility, dispersing agents—typically fetal calf serum, bovine serum albumin or Pluronic F-127—are often used to facilitate cell loading. Pluronic F-127 has also proven useful as a blocking reagent to prevent cell adhesion to PDMS (polydimethylsiloxane, a silicon-based organic polymer) microfluidic channels. We provide Pluronic F-127 in three forms, all of which have low UV absorbance (OD_{280 nm} <0.02 at 10 mg/mL):

• Powder (P6867)
• 10% Solution in water (P6866), filtered through a 0.2 µm–pore size membrane filter for use in tissue culture and other applications
• 20% Solution in DMSO (P3000MP)

Cautioning that Pluronic F-127 is not necessarily physiologically benign, a recent paper shows a Pluronic F-127–dependent modulation of depolarization-evoked Ca²⁺ transients in rat dorsal ganglion (DRG) neurons, as detected with fura-2 AM (F1201, F1221, F1225, F14185; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2).

PowerLoad Concentrate

PowerLoad concentrate (P10020) is 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 available to aid the solubilization of water-insoluble dyes and other materials in physiological media. It is included in the FluxOR Potassium Ion Channel Assay Kits (F10016, F10017; Probes for Ion Channels and Carriers—Section 16.3) to facilitate loading the membrane-permeable AM ester of the FluxOR thallium indicator dye.

We prepare several important reagents for investigating signal transduction, second messenger activity, ion channels and endogenous modulators of intracellular Ca²⁺ concentrations that are described further in Probes for Ion Channels and Carriers—Section 16.3 and Probes for Signal Transduction—Chapter 17.

• Caged cADP ribose (C7074) and 8-amino cADP ribose (A7621)
• Caged D-myo-inositol 1,4,5-triphosphate (NPE-caged Ins 1,4,5-P₃, I23580)
• Thapsigargin (T7458, T7459), BODIPY FL thapsigargin (B7487) and BODIPY TR-X thapsigargin (B13800)
• Fluorescent bisindolylmaleimide analog FIM-1 diacetate (F7453)
• BODIPY FL forskolin (B7469)
• Fluorescent dihydropyridines (D7443, S7445) for L-type Ca²⁺ channels