TEFLabs products in literature, Fluo-2 loaded arteries

  

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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

Background
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.

doi:info:doi/10.1371/journal.pone.0032006.g005

Methodology/Principal Findings
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.

doi:info:doi/10.1371/journal.pone.0032006.g005

Conclusion
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.

doi:info:doi/10.1371/journal.pone.0032006.g005

Calcium fluorescence
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.

doi:info:doi/10.1371/journal.pone.0032006.g005

Figure 5. DCG reduces VSM Ca²⁺ in 4^th-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).

doi:info:doi/10.1371/journal.pone.0032006.g005

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

Fluo-8 Summer Promotion: $50 mg

  

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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

Know your Fluo Indicators

  

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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 vs Fluo-3, Fluo-4, and Fluo-8

  

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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

Procedure:

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

Fluo-2 HA (High Affinity): Excitation at 490 nm, emission at 515 nm

#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

Fluo-2 LA (Low Affinity):  Excitation at 490 nm, emission at 515 nm

#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

Fluo-2 LeakRes (Leakage  Resistant):  Excitation at 490 nm, emission at 515 nm

#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 probes@teflabs.com for bulk quotes.

Fluo-2 MA the brightest fluo indicator

  

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Fluo-2 MA is an ideal indicator for HTS and other calcium applications because of its easy loading, bright response, and lower cost compared to Fluo-3 and Fluo-4.

Fluo-2 MA (U.S. Patent No. 5,049,673) is the same molecule as Fluo-8.

TEFlabs is the lowest cost provider of Fluo-2 and Fluo-8 and can apply aggressive discounts to bulk or frequent orders.

Visit our Fluo-2 page.

Visit our Fluo-8 page.

Fluo-2 variants-HA, LA, LR, NM, MITO, MAG

  

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Fluo-2 HA – High Affinty
Fluo-2 LA – Low Affinity
Fluo-2 LR- Leakage Resistant*
Fluo-2 NM- Near Membrane*
Fluo-2 Mito- Mitochondrial*
Mag-Fluo-2- Magnesium*

Fluo-2 Variant kd's: