At the 17th annual Society of Biomolecular Screening (SBS) conference and exhibition, March 27-31 2011 in Orlando Florida, TEFLabs announced Asante NaTRIUM Green and Asante Potassium Green ion channel dyes to over 1600 scientists from 28 countries.
TEFLabs' new products were one of three winners of the first ever, SLAS New Product Award (NPA) Designations at SBS 2011, which recognized the best of what was new on the exhibit floor. NPA winners were judged by a diverse panel of scientific specialists. The SBS 2011 NPA winners included:
• InSphero: GravityPLUS System
• Pharmadiagnostics: SoPRano Surface Plasmon Resonance (SPR)-Based Screening using a Standard Absorbance Plate Reader
• Teflabs: Asante NaTRIUM Green and Asante Potassium Green Ion Channel Dyes
Three of the most popular long wavelength fluorescent calcium indicators; Fluo-2, Fluo-3, and Fluo-4; were invented by TEF Labs’ creator Akwasi Minta and by Nobel Laureate Roger Tsien at University of California Berkeley (US Patent 5049673, 1991).
Initially Fluo-3 was commercialized because of its low pKa. Fluo-4 became increasingly popular because of its greater "brightness" when excited at the 488 nm argon laser line. (Both Fluo-3 and Fluo-4 are approximately the same "brightness" when excited at their longer wavelength excitation maxima)
At 488 nm, Fluo-2 is the brightest of all, up to twice as bright as Fluo-4,most likely because its AM ester loads more readily into the cell. For their own reasons, other companies have renamed Fluo-2, for example as Fluo-8. In seeking transparency with our customers, we prefer to follow the original nomenclature: Fluo-2.
An experimental comparison of Fluo-2,3,4,and 8 can be found here.
Benefits of Fluo-2 MA:
- A direct substitute for Fluo-3 and Fluo-4
- The same molecule as Fluo-8
- Up to twice as bright as Fluo-4
- Up to four times as bright as Fluo-3
- AM ester loads well at room temperature, faster than Fluo-3 and Fluo-4
- Excitation at 490 nm, emission at 515 nm, and 390 nM Kd
- Greater than two hundred-fold increase from zero to saturated calcium
FLUO-2 MA (AM), US Patent # 5,049,673
(FLUO-2 Medium Affinity, Acetoxymethyl ester)
Fluo-2 Medium Affinity emerges as an ideal candidate over Fluo-3 and Fluo-4 for calcium study. With improved loading characteristics and brightness (nearly 2x as bright as Fluo-4, 4 x as bright as Fluo-3) Fluo-2 MA (the same molecule as Fluo-8) has become a very cost effective replacement or screeners and researchers currently using Fluo-3, Fluo-4 or Fluo-8
Fluo-2 MA, Fluo-3, Fluo-4, Fluo-8 Comparison
REF52 rat embryo fibroblasts
REF52 rat embryo fibroblasts, cultured on No. glass coverslips, were incubated for 45 minutes at room temperature with 5 μM of the AM ester of each indicator in serum-free DMEM containing 25 mM HEPES (pH 7.4). The coverslips were then rinsed in HBSS, mounted in round dishes and bathed with HBSS. Indicators were excited with 488-nm light from a monochromator; fluorescence images were acquired with a cooled CCD camera through a ×40 objective with high numerical aperture (NA = 1.4). Cells were stimulated with 2 μM ionomycin to elicit a sharp rise in intracellular Ca2+ concentration. The images acquired immediately before and after ionomycin addition are shown. The fluorescence change resulting
from ionomycin application in each population of cells was also analyzed; the results are displayed in the bar graph. The result for each indicator is derived from analyzing 35 – 45 cells.
Fluo-2 Products currently available:
Fluo-2 MA (Medium Affinity): Excitation at 490 nm, emission at 515 nm, and 390 nM Kd
TRIAL SIZE #0214 Fluo-2 MA (AM) 2 x 50 ug $15
TRIAL SIZE #0216 Fluo-2 MA (K+SALT) 2 x 50 ug $15
#0200 Fluo-2 MA (AM) 1 mg $110
#0202 Fluo-2 MA (AM) 20 x 50 ug $130
#0204 Fluo-2 MA (K+SALT) 1 mg $110
#0220 Fluo-2 HA (AM) 1mg $110
#0222 Fluo-2 HA (AM) 2 x 50 ug $130
#0224 Fluo-2 HA (K+SALT 1mg $110
#0240 Fluo-2 LA (AM) 1mg $130
#0242 Fluo-2 LA (AM) 2 x 50 ug $150
#0244 Fluo-2 LA (K+SALT) 1mg $130
#0230 Fluo-2 LeakRes (AM) 1mg $130
#0232 Fluo-2 LeakRes (AM) 2 x 50 ug $150
#0234 Fluo-2 LeakRes (K+SALT) 1mg $130
Bulk discounts available, email email@example.com for bulk quotes.
A photomicrograph of cultured embryonic rat hippocampal neurons after 10 days in vitro. The cells were loaded with 3 uM of Asante Calcium Red AM for 30 minutes in culture media then washed in dye-free media for 30 minutes.
Cells were perfused in a flow through chamber and imaged on the stage of an inverted Nikon microscope with a 40 x 1.35 NA objective. Cells were illuminated with Xenon bulb using a 35 nm wide band of light centered around 572 nm and a 60 nm wide band of light centered around 632 nm was imaged with a cooled CCD camera. The scale bar is 20 um.
The below graph shows the percent change in Asante Red Calcium fluorescence in the cell bodies of selected neurons in response to stimulation with NMDA (300 uM) in glycine-containing, Mg-free saline. Images were obtained every 20 seconds. Regions of interest were drawn around the somata, and mean intensities within the regions recorded as a function of time. Percent change of fluorescence at each time was calculated with respect to an average of the first five images.
Images, Graph, and description provided courtesy of J Marks, University of Chicago.
Asante NaTRIUM Green Loading Protocol
The following is supplied as a starting point for non-invasive loading of ANG-1 via the acetoxymethyl (AM) ester version. Due to cell type and other experimental variations, loading conditions will require optimization.
- Prepare a 1 mM stock solution of ANG-1 (AM) in anhydrous dimethylsulfoxide (DMSO).
- For a 500 µg vial, dissolve the contents of the vial in 450 µL DMSO.
- For a 50 µg vial, use 45 µL DMSO.
- 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 ANG-1 (AM) in 50 µg packages.
- Dilute the stock solution to twice the original volume with a solution of 20% Pluronic F-127 in DMSO.
- Disperse the ANG-1/Pluronic F-127 solution into 100 times the volume of serum-free culture medium.
- The final ANG-1 (AM) concentration is 5 µM. (The concentration of ANG-1 AM may require optimization.)
- Pluronic F-127 is 0.1% of the final solution.
- Incubate the cells for one to two hours at room temperature. (Incubation time and temperature may also require optimization.)
- Remove the cell loading medium and wash the cells with serum-free and dye-free medium.
- The cells are now loaded with ANG-1 (AM) and ready for your sodium imaging experiments.
Learn more about Asante NaTRIUM Green-1 and Asante NaTRIUM Green-2.
Download the complete ANG Info Packet HERE
Download the ANG Info Packet HERE!
Asante NaTRIUM Green 1 (ANG-1)
ANG-1 is to Sodium as Fluo-4 is to Calcium
- Compatible with green argon laser, LED excitation, or two-photon IR laser excitation
- Twenty-fold enhancement in emission at 540 nm from zero to saturated sodium
- Sensitive and calibratable response to cytosolic sodium changes
- AM ester loads readily at room temperature
- Little compartmentalization, even distribution throughout the cytosol
- Good retention within cell at 37 °C
- Resistant to photobleaching at 37 °C
- Excitation maximum at 517 nm, emission maximum at 540 nm
- 81 mM Kd for sodium (in the absence of potassium)
- Potassium Salt product specifications
- AM ester product specifications
- Potassium Salt MSDS
- AM ester MSDS
Experimental Results for ANG-1
Excitation and emission spectra of a titration with sodium chloride
Images of ANG-1 response to TRPV1 channel agonization
Images of ANG-1 response to gramicidin-induced sodium increase
Images of ANG-1 response to ouabain inhibition of sodium pump
ANG-1 (AM) Loading Protocols
Acetoxymethyl ester loading protocol for ANG-1
Near-membrane [Ca2+] transients resolved using the
Ca2+ indicator FFP18
ELAINE F. ETTER*, AKWASI MINTAt, MARTIN POENIEt, AND FREDRIC S. FAY*
*Department of Physiology and Biomedical Imaging Group, University of Massachusetts Medical Center, 373 Plantation Street, Worcester, MA 01605;
tTEFLABS, 9503 Capitol View Drive, Austin, TX 78747; and *Department of Zoology, 141 Patterson Laboratories, University of Texas, Austin, TX 78712
Communicated by Joseph F. Hoffman, Yale University, New Haven, CT, December 22, 1995 (received for review August 28, 1995)
ABSTRACT: Ca2+-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Cal] transients recorded using FFP18 during membrane depolarization induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous Ca2+-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.
Download Near-membrane [Ca2+] transients resolved using the
Ca2+ indicator FFP18
FFP-18 is now sold as Fura-2 NearMem (Near Membrane), the molecule remains unchanged.
The following is an excerpt addressing the potential of ACR for multiplexing with Green fluorescent proteins or yellow fluorescent proteins
From a Poster Present at the 2010 SFN conferenece in San Diego, Ca.
Properties of Asante Calcium Red – a novel ratiometric indicator with long excitation wavelengths
Xenia A. Meshik*, Krzysztof L. Hyrc, Mark P. Goldberg**, Washington University School of Medicine, Dept. Neurology,
The Hope Center for Neurological Disorders, Alafi Neuroimaging Laboratory, Saint Louis, MO 63110
"...GFP and YFP, the Green and Yellow Fluorescent Proteins, are commonly used to identify cells successfully modified by genetic manipulations. The presence of YFP and/or GFP makes measurements of [Ca2+]i difficult as the robust protein fluorescence characterized by broad excitation and emission spectra, interferes with the signals of most commonly used indicators. Asante Calcium Red might be less prone to this problem as its emission wavelengths are much longer that those of GFP and YFP (D). To see whether this is the case, we compared changes in the indicator fluorescence in wild-type and GFP expressing neurons (A-C) during depolarization-induced [Ca2+]i elevation (F). The Ca2+- dependent indicator fluorescence (>560 nm) excited at 488 nm, a maximum GFP excitation, rose approximately 1.8 times in GFP-negative and 1.5 times in GFP expressing neurons (F). As the Ca2+-independent indicator emission (500-550) cannot be easily separated from the dominant GFP emission (D), it cannot be used as a reference in GFP/YFP expressing cells. Consequently, in the presence of GFP/YFP, Asante Calcium Red can work only as a single
wavelength indicator. Using different excitation wavelengths in the multi-photon excitation mode is not likely to separate the indicator and GFP fluorescence (E) but might help to isolate GFP-positive cells..."
View or Download the full poster HERE
ORDER ACR HERE
Asante Calcium Red™ offers an alternative to GFP-Certified™ that produces a truly RED emission
Asante Natrium Greeen shares similiar properties to SBFI, shifted into the visible spectrum.
AsanteNatrium Green, TEFLabs' new visible fluorescent sodium indicator, has a selectivity of 1:100 for K+ and the molecule is selective for Na over all other ions.
the selectivity number based on previous results with ghost cells.
Design is based on the fact that there is about 5mM sodium(Na+) in the cell and about 140mM potassium(K+) inside the cell, making selectivity an important issue in experiments done in the presence of potassium.
Asante NaTRIUM Green selects for sodium over potassium about 100 fold, the selectivity holds in the absence of potassium.
Because the selectivity is only relevant in the presence of potassium we found that titration in the presence of potassium selected for sodium over potassium about 20 fold, in three separate studies(normal sodium experiment on confocal microscope, normal sodium ionophore, and an agonist) Asante NaTRIUM Green worked well.
LEARN MORE ABOUT ASANTE NaTRIUM GREEN HERE
A ratiometric ion indicator can work either by “excitation ratioing” or “emission ratioing”.
For excitation ratio, the fluorescence emission at a single wavelength is measured, while excitation alternates between two different wavelengths. For example, this is true for fura-2: emission always measured at ~510 nm while excitation light at 340 nm and 380 nm are delivered alternately. To form the ratio, the emission excited at 340 nm is divided by the emission at 380 nm.
For emission ratioing, fluorescence excitation is done at a single wavelength, while fluorescence emission is measured at two different wavelengths. For example, this is true for indo-1: excitation is often done at ~350-360 nm, while emission is collected at ~410 nm and ~490 nm simultaneously. The ratio is then formed by dividing the 410 emission intensity by the 490 emission intensity.
Whether excitation or emission ratio is used depends on the indicator. In general excitation ratio works when the Ca-free and Ca-bound forms of the indicator absorb light optimally and is thus excited maximally at different wavelengths (e.g., the Ca-free form of Fura-2 has peak excitation at 380 nm, while the Ca-bound form has peak excitation at 340 nm). Irrespective of excitation, however, both forms of the indicator show fluorescence emission that peaks at ~510 nm).
In extremely rare cases (Indo-1 and Asante Calcium Red), the Ca-free and Ca-bound forms actually emit fluorescence at different wavelengths. In these unusual cases, emission ratio can be done.
For More info refer to Methods in Cell Biology, 1994