ION NaTRIUM Green-2 AM Documentation

Buy ION NaTRIUM Green™-2 AM

Back To Top

ION NaTRIUM Green-2 AM Product Specifications

Molecular Weight

1084g/mol

Solubility

DMSO

Kd

20 nM The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. These Kd’s were measured in simple aqueous buffers as a guideline to the scientist, who should then calibrate the dye to his/her system.

Odor

None

Fire and Explosion Hazards

None

Shelf Life

Valid for one year after delivery, if stored properly

TLC

Solvent

5% methanol in chloroform

Rf

0.5

HPLC

Column

C18

Detector Settings

254 nm, 460 nm2

Purity

> 90%

Absorbance Spectrum

Solvent

Methanol

Absorbance max

469 ± 3 nm

ε

18000 M-1cm-1

Fluorescence Spectrum

Solvent

Methanol

Emission max

545 ± 3 nm

Excitation max

525 ± 3 nm

11H NMR

All relevant peaks present

Solvent

Deuterated acetone

Back To Top

ION NaTRIUM Green-2 AM Background

There is a large difference in the sodium ion (Na+) concentration inside and outside the cell (5-40 mM intracellular; 120-450 mM extracellular, depending on organism). This concentration gradient is used to power nutrient uptake, to regulate concentrations of other intracellular ions and solutes, and to generate and transmit electrical impulses in excitable cells such as nerve and muscle. These functions are so important that organisms devote a major part of their metabolic energy to maintaining the sodium gradient. The low intracellular Na+ concentration requires that a Na+ indicator have the sensitivity to measure any small changes that occur. Moreover, intracellular potassium ion (K+) concentration is typically high (in excess of 100 mM); therefore a Na+ indicator should respond selectively to Na+, not K+.

ING-2 is a higher affinity analog of ING-1, as demonstrated by comparing the fluorimetric emission sodium chloride titrations of ING-1 (Figure 1) and ING-2 (Figure 2), with identical excitation and emission wavelengths. It retains the desirable properties of easy loading, slow leakage, and good photostability.

Figure 1

ION Natrium Green-1 Emission Spectra

ION Natrium Green-2 TMA+ Salt Background

Figure 2

ION Natrium Green-2 Emission Spectra

ION Natrium Green-2 TMA+ Salt Background

Back To Top

Ion NaTRIUM Green-2 AM Results

Figure 3

Response of intracellular ING-2 to Na+ influx facilitated by SQI-Pr

REF52 fibroblasts loaded with ING-2 (through incubation with AM ester) were maintained in Hanks’ Balanced Salt Solution (HBSS). SQI-Pr is a Na+ ionophore that promotes Na+/H+ exchange across the cell membrane, and thus causes Na+ influx. Upon application of 40 µM SQI-Pr, the fluorescence of intracellularly-loaded ING-2 increased steadily, reflecting a rise in intracellular [Na+]. Further addition of 20 µM amphotericin B, a polyene natural product that increases membrane permeability to all the common monovalent ions (Na+ K+, H+ and Cl-), gave a small additional increment of indicator fluorescence, as expected. This experiment demonstrates the utility of ING-2 as a sodium indicator, and the efficacy of SQI-Pr as a sodium ionophore. The response shown is the average from 30 cells. [J. Kao, Univ. of Maryland Medical School] ION Natrium Green-2 AM Results

Figure 4

Response of intracellular ING-2 to Na+ influx

REF52 fibroblasts were loaded with ING-2 (through incubation with AM ester) and maintained in 145 mM N-methyl-D-glucamine (NMG) gluconate. To deplete cells of Na+ and K+, the cells were treated with 50 µM amphotericin-B (a polyene microbial metabolite that markedly increases membrane permeability to monovalent ions; e.g., Na+ K+, H+ and Cl-). Increments of NaCl were added to raise sodium concentration ([Na+]) in the extracellular medium to various levels (5, 15, 45, and 145 mM, as indicated in the figure). After each increment, as the intracellular and extracellular [Na+] equilibrated, a corresponding increase in intracellular indicator fluorescence was observed. At the end of the experiment, an aliquot of KCl was added to raise [K+] to 145 mM (typical cytosolic value). The added K+ actually decreased the fluorescence of ING-2 modestly. The response shown is the average from 25 cells. [J. Kao, Univ. of Maryland Medical School] ION Natrium Green-2 AM Results

ING-2 (AM) cell loading procedure for the experiments of Figures 2.6 and 2.7: (This procedure should not be treated as a general loading protocol for ING-2)

(i) 2 µM of ING-2 AM is an average concentration.

(ii) The sample is prepared with ~75 ppm Pluronic for dispersing the dye into the incubator buffer.

(iii) The buffer consisted of HCO3- DMEM with 10% fetal bovine serum (FBS) and kept under 5% CO2 atmosphere.

(iv) Loading time varied from 45 minutes to 70 minutes.

(v) The experiments were conducted in HBSS.

Figure 2.6

ION Natrium Green-2 AM Results

Figure 2.7

ION Natrium Green-2 AM Results [J. Kao of Univ. of Maryland Medical School]

Back To Top

Ion NaTRIUM Green-2 AM Experimental Methods

Figure 3 Methods

REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM ION Natrium Green-2 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic surfactant, Pluoronic F-127. The cells were then transferred into HBSS. SQI-Pr was prepared as a 20 mM stock solution in DMSO; amphotericin B was prepared as a 50 mM solution in DMSO. Appropriate volumes of the stock solutions were added to the HBSS bathing the cells to achieve the desired final concentrations used in the experiment. Indicator fluorescence was excited at 488 nm; fluorescence images were acquired with a cooled CCD camera. [J. Kao, Univ. of Maryland Medical School]

Figure 4 Methods

REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM ION Natrium Green-2 AM ester in bicarbonate-buffered DMEM, supplemented with 10% fetal bovine serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic surfactant, Pluoronic F-127. The cells were then transferred into 145 mM NMG gluconate (pH 7.4). Amphotericin B was prepared as a 50 mM solution in DMSO; 1000-fold dilution into aqueous medium gave the desired final concentration used in the experiment. Appropriate volumes of 4 M NaCl or KCl were added to achieved the desired concentrations in the aqueous medium. Indicator fluorescence was excited at 488 nm; fluorescence images were acquired with a cooled CCD camera. [J. Kao, Univ. of Maryland Medical School]

ING-2 Response to Na+ Influx Facilitated by SQI-Pr

Plot quantifying the enhanced fluorescence of ING-2, resulting from increased cytosolic sodium ion concentration provoked by the application of the sodium ionophore SQI-Pr. REF52 fibroblasts loaded with ING-2 (AM) in HBSS were exposed to 40 μM SQI-Pr, a Na+ ionophore that promotes Na+/H+ exchange across the cell membrane. The fluorescence of intracellular ING-2 increased steadily, reflecting a rise in intracellular [Na+]. Further addition of 20 μM amphotericin B, a polyene natural product that increases membrane permeability to all the common monovalent ions, gave a small additional increment of indicator fluorescence, as expected. This experiment demonstrates the utility of ING-2 as a sodium indicator and the efficacy of SQI-Pr as a sodium ionophore. The response shown is an average from 30 cells. Experimental results courtesy of Dr. JPY Kao, University of Maryland

ION Natrium Green-2 AM Experimental Methods

ING-2 Intracellular Titration with NaCl

Plot quantifying the enhanced fluorescence of ING-2, resulting from increased cytosolic sodium ion concentration. REF52 fibroblasts loaded with ING-2 (AM) were maintained in 145 mM N-methyl-D-glucamine (NMG) gluconate. To deplete cells of Na+ and K+, the cells were treated with 50 μM amphotericin B, a polyene natural product that increases membrane permeability to all the common monovalent ions. Increments of NaCl were added to raise the sodium concentration in the extracellular medium to various levels (5, 15, 45,and 145 mM). After each increment, as the intracellular and extracellular [Na+] equilibrated, a corresponding increase in intracellular indicator fluorescence was observed. At the end of the experiment, an aliquot of KCl was added to raise [K+] to 145 mM. The added K+ decreased the fluorescence of ING-2 modestly. The response shown is an average from 25 cells.

Experimental results courtesy of Dr. JPY Kao, University of Maryland

ION Natrium Green-2 AM Experimental Methods

ING-2 Response to Voltage-Gated Sodium Channel Stimulation

NaV1.3-expressing HEK-293 cells loaded with ING-2 (AM) were exposed to a concentration series of lidocaine, a voltage-gated sodium channel blocker. The cells were stimulated with 25 mM veratridine in 20 mM HHBS.

Experimental results courtesy of Dr. Dave Weaver, Vanderbilt University

ION Natrium Green-2 AM Experimental Methods

Effect of Excitation Wavelength on ING-2 Emission Intensity

Emission spectra for a 150 mM NaCl solution of ING-2 (TMA+ Salt) in 130 mM TMACl, 10 mM MOPS, pH 7.1 at 488, 505, 510, and 517 nm excitation. Emission intensity drops to nearly a third of that at optimal (517 nm) excitation by exciting at 488 nm.

ION Natrium Green-2 AM Experimental Methods

Back To Top

Ion NaTRIUM Green-2 AM Loading Protocol

A sample loading procedure may be accessed in Merck Research Laboratories’ SLAS 2011 poster by O’Donnell, et al., for ING-2 in a 1536-well voltage-gated sodium channel assay.

The following is supplied as a starting point for non-invasive loading of ING-2 via the acetoxymethyl (AM) ester version. Due to cell type and other experimental variations, loading conditions will require optimization.

  1. Prepare a 1 mM stock solution of ING-2 AM in anhydrous dimethylsulfoxide (DMSO). (A more concentrated solution may be used to minimize final DMSO percentage.)
    • For a 500 μg vial, dissolve the contents of the vial in 460 μL DMSO.
    • For a 50 μg vial, use 46 μL DMSO.
  2. For a 500μg vial, divide the stock solution into aliquots that will be consumed per experiment.
    • Store the aliquots at -20 ºC, protected from moisture and light.
    • Repeated freezing and thawing of a stock solution typically leads to degradation of the product via hydrolysis of the AM esters
    • For your convenience, we supply ING-2 (AM) in 50 μg packages.
  3. Dilute the stock solution to twice the original volume with a solution of 20% Pluronic F-127 in DMSO.
  4. Disperse the ING-2/Pluronic F-127 solution into 100 times the volume of serumfree culture medium.
    • The final ING-2 (AM) concentration is 5 μM. (The concentration of ING-2 AM may require optimization.)
    • Pluronic F-127 is 0.1% of the final solution. (The final concentration of Pluronic F-127 may require optimization.)
  5. Incubate the cells for one hour at room temperature. (Incubation time and temperature, 20°C vs. 37°C, may also require optimization.)
  6. Remove the cell loading medium and wash the cells with serum-free and dyefree medium.
  7. The cells are now loaded with ING-2 AM and ready for your sodium imaging experiments.

Back To Top