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

Page Contents

• Caged Calcium and Caged Calcium Chelators
• Nonfluorescent Chelators
• Calcium Calibration Buffer Kits
• Influx Pinocytic Cell-Loading Reagent
• Loading P2X7 Receptor–Expressing Cells
• Ionophores
• Probenecid: Inhibitor of Organic-Anion Transporters
• Pluronic F-127 and PowerLoad Solutions
• Data Table
• Ordering Information

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). Other suitable ultraviolet light sources for photoactivation of NP-EGTA include mercury arc lamps (365 nm), frequency-tripled Nd:YAG lasers (355 nm), UV argon-ion laser lines (351/364 nm) and light-emitting diodes (LED). NP-EGTA is not suitable for two-photon photoactivation due to its extremely low absorption cross section in the wavelength range accessible with femtosecond pulsed Ti:sapphire laser sources.

We offer the tetrapotassium salt (N6802) 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 19.8.1). 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), 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 19.8.2). 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⁺. Unlike NP-EGTA, DMNP-EDTA has a low but finite two-photon absorption cross-section in the wavelength range accessible with femtosecond pulsed Ti:sapphire laser sources  and can therefore be used to produce two-photon activated calcium release with high spatial precision (Figure 19.8.3). Two reviews by Ellis-Davies discuss the uses and limitations of DMNP-EDTA.

Diazo-2: A Photoactivatable Calcium Knockdown Reagent

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 is a photoactivatable Ca²⁺ scavenger. Diazo-2, 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). 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. Intracellular loading of NP-EGTA, DMNP-EDTA and diazo-2 is best accomplished by patch pipette infusion with the carboxylate salt form of the caged compound added to the internal pipette solution at 1–10 mM.

BAPTA and BAPTA AM

The bis(2-aminophenoxy)ethane tetraacetic acid (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 19.8.4). 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 AM (or the higher-affinity 5,5’-dimethyl BAPTA AM) is also useful for establishing an intracellular zero free calcium level for in situ calibrations of fluorescent indicators (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). BAPTA and its derivatives also exert physiological effects that are somewhat independent of their calcium binding activity.

BAPTA is available as a cell-impermeant potassium, cesium or sodium salt (B1204, B1212, contact Custom Services for more information); the Cs⁺ salt of BAPTA has frequently been used for patch-clamp experiments. In addition, we offer the cell-permeant BAPTA AM ester in two packaging formats (B1205, B6769).

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.

BAPTA derivatives with intermediate affinity for Ca²⁺, such as 5,5'-dibromo BAPTA, have been extensively used to study Ca²⁺ mobilization, spatial Ca²⁺ buffering and Ca²⁺ shuttling in a variety of cells and animal models. 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, have been employed for optical imaging studies  but are most widely used for NMR analysis of Ca²⁺ in live cells and tissues. 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.

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.

DTPA Isothiocyanate

DTPA isothiocyanate can be coupled to antibodies and other biomolecules by conventional amine-reactive chemistry, thereby introducing a high-affinity binding site for lanthanides and other 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 and ligands labeled with the fluorescent lanthanides europium and terbium are widely utilized in time-resolved fluorescence–based assays. 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

A calcium sponge polymer is 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. The 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. 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 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 kits designed to facilitate this calibration using a laboratory fluorometer or quantitative imaging system. 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 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 Kit #1

Because cells contain very low levels of free Ca²⁺, it is essential to use Ca²⁺ buffers such as EGTA to precisely calibrate Ca²⁺ indicators under specific 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.

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 cross-mixed to give a series of solutions with equal dye and varying free Ca²⁺ concentrations. With ratiometric indicators, this method yields a series of curves that exhibit an accurate isosbestic point (see, for example, (Figure 19.8.5); it is the method regularly used in our laboratories to determine Ca²⁺ affinities. In situ calibrations of intracellular calcium indicators can be carried out using CaEGTA buffer solutions in combination with a suitable ionophore such as ionomycin (I24222, see below).

Figure 19.8.5 Fluorescence excitation spectra of fura-2 (F1200, F6799) in solutions containing 0–39.8 µM free Ca2+.

Fura-2 Calcium Imaging Calibration Kit

The Fura-2 Calcium Imaging Calibration Kit (F6774) is designed to facilitate rapid calibration and standardization of digital imaging microscopes. This kit provides 11 CaEGTA:EGTA buffer solutions with free Ca²⁺ concentrations from zero to 40 µM. Each solution also includes 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 19.8.6).

The Influx pinocytic cell-loading reagent is 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, DNA, siRNAs, antisense oligonucleotides and Qdot nanocrystals —into a variety of cell lines.

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 (C14784, Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) facilitates cell loading and slide handling when using the Influx reagent.

Figure 19.8.6 Principle of the Influx reagent pinocytic cell-loading method (I14402). Cultured cells are placed in hypertonic Influx loading reagent (panel A), along with the material to be loaded into the cells (yellow fluid, panel B), allowing the material to be carried into the cells via pinocytic vesicles. When the cells are placed in hypotonic medium, the pinocytic vesicles burst (panel C), releasing their contents into the cytosol (panel D).

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
  
• Aqueous tracers: lucifer yellow CH (L453, L1177; Polar Tracers—Section 14.3) and sulforhodamine B (S1307, Polar Tracers—Section 14.3)
• Nucleic acid stain: YO-PRO-1 (Y3603, Nucleic Acid Stains—Section 8.1)

BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate is one of the most potent and widely used P2X receptor agonists, . 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 19.8.7) 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, Figure 19.8.7), 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 Mg2+ Indicators—Section 19.6). 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).

Figure 19.8.7 Chemical structures of the ionophores A-23187 (R = H, A1493) and 4-bromo A-23187 (R = Br).

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. The use of probenecid to block the efflux of intracellular dyes was first reported by Di Virgilio and co-workers. Wash steps or masking 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. Masking dyes, while offering the advantage of homogeneous (one-step, mix-and-read) assays, may interact negatively with some receptor systems of interest. Our water-soluble probenecid (P36400) has the advantage of being easy to dissolve in physiological buffers, unlike the conventionally used free acid form, which must be initially dissolved in strong base.

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 surfactants that act to disperse and stabilize water-insoluble dyes in aqueous solution, thereby facilitating cellular uptake from aqueous dispersions. The PowerLoad reagent is supplied as a ready-to-use concentrate in sterile water that is mixed with the dye stock solution (typically prepared in DMSO) and then diluted 1:100 into serum-free medium for direct application to adherent cells on coverslips, in microplate wells or in other suitable incubation chambers (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1).

For a detailed explanation of column headings, see Definitions of Data Table Contents