Asante NaTRIUM Green-2

 It is not recommended to store ANG-2 in DMSO for long periods of time, even at -20C. If you plan on storing unused ANG-2 for later use we recommend breaking down and storing ANG-2 into aliquots before the addition of DMSO.  TEFLabs offers a pre-aliquoted 10 x 50 µg ANG-2 (#3512)

Asante NaTRIUM Green-2 (ANG-2) is a higher affinity analog of ANG-1, as demonstrated by comparing the fluorimetric emission sodium chloride titrations of ANG-1 (Figure 2.4) and ANG-2 (Figure  2.5), with identical excitation and emission wavelengths.  It retains the desirable properties of easy loading, slow leakage, and good photostability.

Publication:

A recent article by Christophe Lamy, and Jean-Yves Chatton highlights the uses and improved performance of TEFLabs' novel sodium indicator, Asante NaTRIUM Green™

Christophe M. Lamy, Jean-Yves Chatton, Optical probing of sodium dynamics in neurons and astrocytes, NeuroImage, Volume 58, Issue 2, 15 September 2011, Pages 572-578, ISSN 1053-8119, 10.1016/j.neuroimage.2011.06.074.

 

View the MERCK poster presented at SLAS 2012:

Evaluation of the Sodium Sensing Dye Asante Natrium Green 2 in a Voltage-gated Sodium Channel Assay in 1536-well Format

Gregory T. O'Donnell, Kelli Solly, Carissa Quinn, Brian Squadroni, Eric Johnson, Jeffrey Hermes, and Michael Finley
Department of Exploratory Sciences and Screening
Merck Research Laboratories 140-154 Wissahickon Ave, North Wales, PA 19454, USA

DATA

Excitation: 488 - 517 nm (excitation maximum at 517 nm)

Emission: 540 nm

kd: 20 mM

Solubility: H2O (salt form), DMSO (AM form)

Molecular Weight: 1100 g/mol

 

FIG. 2.4 Asante Natrium Green 2 Emission Spectra

Asante NaTRIUM Green 2 Emission Titration

 

 

FIG. 2.5 Asante Natrium Green 1 Emission Spectra

ANG-1 fluorescence spectra

 

RESULTS

Two example applications of ANG-2 in cells are shown in Figures 2.6 and 2.7 below:

ANG 2 resized 600 
Figure 2.6 Response of intracellular ANG-2 to Na+ influx facilitated by SQI-Pr:  REF52 fibroblasts loaded with ANG-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 ANG-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 ANG-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]

 

 

 

 

fig 2.7 ang 2 resized 600Figure 2.7 Response of intracellular ANG-2 to Na+ influx: REF52 fibroblasts were loaded with ANG-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 ANG-2 modestly. The response shown is the average from 25 cells. [J. Kao, Univ. of Maryland Medical School]

 

ANG-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 ANG-2)

(i) 2 µM of ANG-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.

[J. Kao of Univ. of Maryland Medical School]

 

 

 

Experimental Methods

Figure 2.6 Methods: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante 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 2.7 Methods: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante 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 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 4.0 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]