Asante Potassium Green

Asante Potassium Green is a fluorescent indicator with a useful Kd for measuring cytosolic K+ concentration. It loads readily and is excited by visible light. Although non-ratiometric, its large fluorescence dynamic range allows sensing of even small changes in K+ concentration. Optimal excitation occurs at 517 nm, but the indicator can also be excited at the conventional wavelength of 488 nm. APG-1 works well with 2-photon excitation at near-infrared wavelengths and is quite resistant to photobleaching. Like its sodium counterpart, it is useful for confocal microscopy, flow cytometry, and screening.  Asante Potassium Green-1 has been replaced by our Asante Potassium Green-2 with improved kd.

DATA

Excitation: 488 nm (excitation maxima at 517 nm)
Emission: 540 nm
kd: 54 mM
Solubility: H2O (salt form),  DMSO (AM form)
Molecular Weight: 1100 g/mol


RESULTS

 

Asante Potassium Green 1 emission spectra

Asante Potassium Green 1 emission titrattion in TMA+CL-.


Response of intracellular Asante Potassium Green to K+

Figure 3.1 Response of intracellular APG-1 to K+:  REF52 fibroblasts loaded with APG-1 (through incubation with AM ester) were maintained in 145 mM N-methyl-D-glucamine (NMG) gluconate. To deplete cells of K+ and Na+, 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 KCl were added to raise potassium concentration ([K+]) 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 [K+] equilibrated, a corresponding increase in intracellular indicator fluorescence was observed. At the end of the experiment, an aliquot of NaCl was added to raise [Na+] to 10 mM, a typical cytosolic value. The added Na+ only slightly affected the fluorescence of APG-1. The response shown is the average from 45 cells. [J. Kao, Univ. of Maryland Medical School]

 

APG-1 (AM) cell loading procedure for the experiment of Figures 3.1: (This procedure should not be treated as a general loading protocol for APG-1. Please refer to our AM Ester Loading guidelines.)

  1.  1-5 μM of APG-1 (AM) is typical concentration range for loading
  2. The sample Is prepared with ~75 ppm Pluronic for dispersing the dye into the incubation buffer.
  3. For these experiments, the buffer consisted of HCO3-DMEM with 10% fetal bovine serum (FBS) and kept under 5% CO2 atmosphere.
  4. Loading time varied from 45 minutes to 70 minutes.
  5. Fluorescence imaging experiments were conducted in HBSS.

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

 

 

Figure 3.1 Methods:  REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante Potassium Green 1 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; 1,000-fold dilution into the aqueous medium gave the desired final concentration of 50 µM. Appropriate volumes of 4.0 M KCl or NaCl were added to achieve 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]

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Asante Potassium Green-2

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