SLAS 2013 Poster Alert:
ANG-2 compared to elecrophisiology
Natsue Yamanada of Shionogi & CO., Ltd. presents a comparison of ANG-2 (TEFLabs) and IonWorks Barracuda (Molecular Devices, Inc.) in voltage-gated sodium channels.
Natsue Yamanada – Shionogi & CO., Ltd.
Date: 1/15/2013 Time: 1:00 PM - 3:00 PM
See ThallosTM in action at SLAS 2013. Visit TP 165 Tuesday Jan. 15th from 1-3pm :
Date: 1/15/2013 Time: 1:00 PM - 3:00 PM TP 165
Using “no wash” format Thallos produces a higher fold increase in fluorescence compared to FluxOR Tl+ indicator. Both dyes produced excellent Z’ (ThallosTM = 0.87, FluxORTM Tl+ indicator = 0.68) in this assay and nearly identical potencies for compounds tested.1
1 Image and description courtesy of Dave Weaver, Vanderbilt University. Sudies performed in mammalian potassium channel.
BCECF is the most widely used fluorescent pH sensor, its pKa (6.98) is near physiological pH and allows the detection of cytosolic pH change with high sensitivity. The excitation spectrum of the dye undergoes a slight shift during pH change, while the wavelength of the emission maximum remains unchanged. The pH is determined ratiometrically by the relative fluorescent intensities at 535 nm when the dye is excited at 439 nm and 505 nm respectively. At low pH, BCECF is weakly fluorescent, however, fluorescence increases with increasing pH.
Generic loading protocol for BCECF:
mammalian cells can be loaded by incubation with the membrane permeant
BCECF AM. Inside the cell, nonspecific esterases hydrolyze the nonfluorescent AM ester into
the fluorescent, pH-sensitive indicator. The low
leakage rate of the negatively charged BCECF and the small intracellular
volume results in the higher intracellular concentration compared to the extral incubation concentration. BCECF is typically less susceptible to intracellular compartmentalization than calcium indicators.
1. Prepare cells in suspension
2. Dilute a1 mM aliquot of AM ester stock solution 100-500 fold into a physiological saline buffer (HBSS for example). It is recommended to use the minimum concentration of AM ester necessary to obtain an adequate signal (0.1 μM may be sufficient). The loading medium needs to be free of amino acids or buffers containing amines, to ensure there is no interference with hydrolosis of AM esters.
3. Add one-one volumes of aqueous AM ester dispersion and cell suspension. Incubate for 15–60 minutes at 4°C to 37°C.
4. Wash the cells with fresh culture medium 1-3 times.
The general protocol has also been aplied to tisue samples such as rat arteires and salivary glands, rabbit kidney and gastric glands. Typical applications involves mounting the tissue sample in a perfusion chamber and adding 1-5μM of BCECF to the perfusate for between 10-70 minutes. An wash withunmodified perfusate is then performed.
Learn more about BCECF
A group from Merck's Department of Exploratory Sciences and Screening presented a poster evaluating Asante Natrium Green-2 in a 1536 well voltage -gated sodium channel Assay.
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
High-throughput screening (HTS) of voltage-gated sodium channels has required the use of indirect measurements of channel activity partly due to the lack of a robust sodium sensitive dye. One popular screening method utilizes a dye that responds to changes in the membrane potential that results from sodium channel opening. Another approach, atomic absorption spectroscopy, makes use of surrogate ion transport through the voltage-gated ion channel of interest, substituting lithium for sodium. While these assays have been shown to be amenable to high-throughput screening, the direct measurement of sodium flux in an HTS-friendly read-out would be beneficial in lead identification efforts. Herein we describe a 1536-well FLIPR screening assay for antagonists to a voltage-gated sodium channel expressed in human embryonic kidney cells (HEK293) using a sodium sensing dye, Asante Natrium Green 2 (ANG-2, Teflabs). Addition of 60 μM of the site 2 agonist veratridine induced a signal-to-background ratio (S/B) of 1.3-1.6 fold (control wells versus wells treated with an IC100 of tetracaine). The assay was benchmarked against three known voltage-gated sodium channel blockers, tetracaine, flecainide, and mexilitene and exhibited acceptable sensitivity with IC50s of 2.7, 15.3, and 29 uM, respectively. The assay was then used to screen a library of 27,978 small molecules to assess the performance under screening conditions and compared to results using the same compound set screened with a membrane potential dye (Blue component A, Molecular Devices). The sodium dye assay gave robust statistics with Z' values averaging 0.71 and S/B averaging 1.58-fold over a 48 plate screening run. While the sodium dye showed good internal consistency (R2 = 0.80 when plotting duplicate data for each compound against each other), the membrane potential and sodium dye assays showed a weaker correlation with an R2 = 0.46. Using a 40% activity cut-off, 2237 compounds showed overlapping activity in both assays. However, each assay identified a large number of unique hits with 886 sodium dye only hits and 1186 membrane potential dye only hits. Data on the same compounds obtained from the IonWorks Quattro (Molecular Devices) system revealed that the sodium dye identified 39.3 % of all the electrophysiology positives, while the membrane potential dye identified 39.4 %. However, the sodium dye detected 129 electrophysiology actives that the membrane potential dye missed, while the membrane potential dye identified 130 compounds missed in the sodium dye assay. While neither assay appears capable of identifying all the potential inhibitors that may be uncovered using an electrophysiology read-out, both dyes provided robust read-outs in 1536-well format and would allow for the screening of large compound libraries. Additionally, the use of both membrane potential and sodium dyes in an HTS strategy may prove beneficial as both identify unique hits.
A total of 27,978 compounds were screened in the three different assays. The Venn diagram shows the hit distribution and overlap of those hits. Although the overall hit rates at 40% inhibition or greater for the sodium and membrane potential dye assays were similar (13.5% and 14.6%, respectively), each assay identified large numbers of assay specific hits. Combining the sodium and membrane potential assay hits, only 47% of all the electrophysiology hits were identified in the FLIPR screens.
Congratulations to Sandeep Khurana M.B.B.S., Hema Raina M.B.B.S., M.D., Ph.D., Valeria Pappas Ph.D., Jean-Pierre Raufman M.D., and Thomas L Pallone M.D., for their recent publication:
Khurana S, Raina H, Pappas V, Raufman J-P, Pallone TL (2012) Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries. PLoS ONE 7(2): e32006. doi:10.1371/journal.pone.0032006
Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.
Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K+ channel blockade with glibenclamide (KATP), 4-aminopyradine (KV), BaCl2 (KIR) or tetraethylammonium (TEA, KCa) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca2+ fluorescence (~50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca2+ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca2+ responses (~50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.
In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca2+ responses, independent of muscarinic receptor, NO or K+ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.
In experiments where a Ca2+ indicator was used, the selected artery was exposed to dissection solution containing Fluo-2 (TEFLabs, 7.5 µM), 1.5% DMSO (vol/vol), and 0.03% cremophor EL (vol/vol) for 1 h at room temperature. After 1 h, the arteries were cannulated as described above and allowed to develop MT for 30 min. Arteries were imaged with a confocal scanning inverted microscope (×60, 1.4 NA, water-immersion objective). Images of fluo-2-loaded fluorescent VSM were obtained with an intensified CCD camera (Stanford Photonics, Palo Alto, CA, USA) coupled to a Nipkow spinning disk confocal microscope with 488 nm excitation. Spatially resolved information on cytoplasmic [Ca2+] was obtained in individual VSM cells. To quantify changes, fluo-2 fluorescence (F) was normalized to its initial value (F0) in each cell.
Figure 5. DCG reduces VSM Ca2+ in 4th-order mesenteric arteries from rats with MT.
Ca2+ fluorescence was measured in Fluo-2-loaded arteries before and after incubating with DCG 100 µM for 5 minutes. DCG reduced the arterial VSM Ca2+ fluorescence by ~50%. (n = 3 arteries in each group).
Copyright Khurana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries
A preliminary response of Asante Natrium Green-2 with sodium channel receptor, Nav 1.3 expressed in HEK 293 cells is shown in Figure 1 below. The wavelength of excitation used was 488 nm. Figure 2 shows various excitation wavelengths including emission maxima 517 nm. Filters or Flexstation may be neccessary for maximum response.
Figure 1: Na+ flux in NaV 1.3-expressing HEK-293 cells loaded with ANG-2 and treated with a concentration series of lidocaine. Cells stimulated with 25 mM vertradine in 20 mM HEPES-buffered HBSS.
Figure 2 (below): The maximum emission reached on ANG-2TMA+ at different excitation Wavelengths.
Rhod-2, with fluorescence excitation maxima at 552 nm and emission maxima at 581 nm, was first introduced in 1989. Rhod-2 is essentially non-fluorescent before Ca2+ binding, becoming more fluorescent with increasing Ca2+ concentration. The longer excitations and emissions of Rhod-2 make the indicator useful for experiments in cells and tissues that have high levels of autofluorescence and for multiplexing with other fluorescent dyes of shorter wavelengths.
TEFLabs offers high Quality Rhod-2 (>90% by HPLC) at significant savings compared to other Rhod-2 suppliers:
Learn more about TEFLabs Rhod-2 pricing and availability
Rhod-2 pricing (current as of 20 july 2011):
TEFLabs 1mg $110
Invitrogen 1mg $ 319
Anaspec 1mg $247.50
Enzo 1mg $239
AAT 1mg $175
Biotium 1mg $138
Learn more about TEFLabs Rhod-2 pricing and availability
A recent article by Christophe Lamy, and Jean-Yves Chatton highlights the uses and improved performance of TEFLabs' novel sodium indicator, Asante NaTRIUM Green-1™, over previous fluorescent sodium indicators like Sodium Green™, CoroNa Green™, and CoroNa Red™.
Sodium Green™, CoroNa Green™, and CoroNa Red™ are registered trademarks of Life Technologies, Inc.
TEFlabs has the lowest prices ever for Fluo-8. Beat the heat this summer, spend more time indoors performing experiments using Fluo-8. (expires 10-1-2011)
#0201 TEFlabs Fluo-8 (AM) 1 mg $50
#0203 TEFlabs Fluo-8 (AM) 20 x 50 ug $60
#0205 TEFlabs Fluo-8 (K+Salt) 1 mg $50
While Fluo-8 is marketed under a variety of names, the original patent outlining Fluo-2 medium affinity (Fluo-8) published in 1991. The original journal publication appeared in the Journal of Biological Chemistry in 1989. Why pay a premium for 20 year old indicator technology?
Some companies will charge $245 or more per mg, TEFLabs has been making Fluo dyes since 1988 and has more Fluo experience than any of our competitors, our experience and expertise at manufacturing Fluo dyes allows us to offer Fluo-8 at much lower cost to researchers. Dont let other manufacturers inexperience cost you money!
Read the original Fluo paper:
Fluorescent Indicators for Cytosolic Calcium based on Rhodamine and Fluorescein Chromophores
TEFLabs Inventory Reserve program allows customers to get favorable per unit pricing based on anticipated annual consumption. TEFLabs will hold a specified amount of inventory for customers who can then order throughout the year from their reserved inventory. Customers get the price benefit of a one time bulk order but only pay for products as they are used.
In addition to the best possible volume discounts the Inventory Reserve program allows researchers to receive all their reserved inventory from a single lot, guarantees the immediate availability of inventory, and allows researchers to pay for products as they are ordered without having to purchase up front and store in-house.
TEFLabs Inventory Reserve program can be used for annual orders of 10 units up to multi gram scale. Inventory Reserve orders typically receive higher bulk rate discounts than TEFlabs advertised quantity discounts.
To request an Inventory Reserve quote simply complete and return the form below:
TEFLabs Inventory Reserve Order Form