Download Red calcium indicator

FluoProbes®
FT-FJ2970
Asante Calcium Red
Red ratiometric calcium indicator
Product Description
Name :
Asante Calcium Red, K+ salt
Catalog Number :
FP-FJ2980, 250 µg
Molecular Weight :
Kd :
Pka :
Solubility:
Absorption / Emission :
MW= ~1000 g/mol
~300-400 nM
~5.5
EC (M-1 cm-1) :
Methanol
λexc\λem = 540 / 650 nm for non-ratiometric assay
λexc\λem = 488 / 517, 650 nm for ratiometric assay
45000 M-1cm-1
Name :
Asante Calcium Red, AM
Catalog Number :
FJ2970, 500 µg FJ2971, 10 x 50 µg
Molecular Weight :
Solubility:
Absorption / Emission :
MW= ~1200 g/mol
DMSO
See K+ salt for spectral characteristics after AM group cleavage
Storage:
-20°C
Protect from light and moisture.
Introduction
Asante is a new family of fluorescent ion indicator dyes that offer researchers maximum flexibility, reliability
and ease of use.
Asante's revolutionary compound combines the best qualities of UV and non-UV dyes with new features that
make it ideal for a wide range of research and testing:
emission ratiometric mode
• Single excitation at argon laser 488 nm, dual emission at 520 nm and 650 nm
• Greater than twenty fold emission enhancement at 650 nm and decrease to about half of emission at 520
nm, upon saturating with calcium
• Ratiometry eliminates variables such as dye concentration, optical path, and instrumental variations to
give an accurate calibratable reading.
• NOT hydrophobic like Fura-Red
• FULLY longwave visible, unlike Fura-Red and BTC
• NOT a dual fluorophore, artificially ratiometric system, such as Fluo with SNARF
emission non-ratiometric mode
•
•
•
•
Excitation 540 nm, calcium dependent emission enhancement at 650 nm (Stokes shift 110 nm)
Greater than fifty fold emission enhancement upon saturating with calcium
Longer wavelength eliminates cellular autofluorescence encountered with Fluo indicators.
Virtually nonfluorescent in the absence of calcium, unlike the longer wavelength Rhod dyes
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Instructions for use
–
–
–
Stock solution in DMSO
Incubate cells in 3 to 10 µM Asante Calcium Red (AM) and 0,02% Pluronic F-127 for at least 45
minutes at room temperature
Wash cells and maintain in medium for at least another 45 minutes to allow complete hydrolysis of AM
esters by esterases
Specifications
Absorption Spectrum
Solvent
Methanol
λmax
555 nm
ε
45000 M-1cm-1
Excitation Spectra
(Figure 1)
Solvent
10mM EGTA, 100mM KCl, 10mM MOPS, pH 7.2
λem
650 nm
λmax
540 nm
Non-Ratiometric
Emission Spectra
(Figure 2)
Solvent
10mM EGTA, 100mM KCl, 10mM MOPS, pH 7.2
λex
540 nm
λmax
650 nm (emission intensity increases strongly with increasing calcium)
Ratiometric Emission Spectra (Figures 3 and 4)
Solvent
10mM EGTA, 100mM KCl, 10mM MOPS, pH 7.2
λex
488 nm
λmax
517 nm (emission intensity decreases slightly with increasing calcium)
650 nm (emission intensity increases strongly with increasing calcium)
All titrations were performed with varying proportions of 10 mM K2EGTA, 100 mM KCl, 10 mM MOPS and 10
mM CaEGTA, 100 mM KCl, 10 mM MOPS at pH 7.2
Figure 1: Excitation traces of a calcium titration of Asante Calcium Red (K+ Salt)
sfc (K+ Salt) lot 910a
fluorescence intensity (cps)
3500000
emission at 650 nm
3000000
2500000
2000000
1500000
1000000
500000
0
450
500
550
excitation wavelength (nm)
600
0 nM Ca2+
17 nM Ca2+
38 nM Ca2+
65 nM Ca2+
100 nM Ca2+
225 nM Ca2+
351 nM Ca2+
602 nM Ca2+
1350 nM Ca2+
39000 nM Ca2+
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150 nM Ca2+
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Figure 2: Non-ratiometric emission traces of a calcium titration of Asante Calcium
Red (K+ Salt)
sfc (K+ Salt) lot 910a
excitation at 540 nm
fluorescence intensity (cps)
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
550
600
650
700
750
emission wavelength (nm)
0 nM Ca2+
225 nM Ca2+
17 nM Ca2+
351 nM Ca2+
38 nM Ca2+
602 nM Ca2+
65 nM Ca2+
1350 nM Ca2+
100 nM Ca2+
39000 nM Ca2+
150 nM Ca2+
Figure 3: Ratiometric emission traces of a calcium titration of Asante Calcium Red
(K+ Salt), lot 910a
sfc (K+ Salt) lot 910a
excitation at 488 nm
fluorescence intensity (cps)
2100000
1400000
700000
0
500
550
600
650
700
750
emission wavelength (nm)
0 nM Ca2+
150 nM Ca2+
17 nM Ca2+
225 nM Ca2+
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38 nM Ca2+
351 nM Ca2+
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39000 nM Ca2+
100 nM Ca2+
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Figure 4: Ratiometric emission traces of a calcium titration of Asante Calcium Red
(K+ Salt), lot 908a
fluorescence intensity (cps)
900000
sfc (K+ Salt) lot 908a
excitation at 488 nm
600000
300000
0
500
550
600
650
700
wavelength (nm)
0 nM Ca2+
50 nM Ca2+
150 nM Ca2+
450 nM Ca2+
39000 nM Ca2+
Note: Calibration of sfc (K+ Salt) in the cell has not been performed yet. Initial results in rat cardiac myocytes
and neurons show an attenuated response at ~520 nm, with little to no response to calcium changes. In contrast,
calcium changes produce a strong response at 650 nm. In our hands, as can be noted from the variance in the
lots 908a and 910a, we have not yet achieved consistent results for the 517 nm peak, although the 650 nm peak
has consistently shown a ~20-fold increase in emission upon calcium saturation.
Note2: Exciting at 488 nm produces only 40% of the emission at 650 nm obtained from 540 nm excitation
(Figure 1), so that adjustments for the reduced emission (i.e., higher dye concentration or increasing power to
the excitation source) may be necessary.
Emission ratiometric imaging of ATP-evoked Ca2+ transient in rat vagal sensory neuron
Neurons
The inferior vagal (nodose) ganglia from a Sprague-Dawley rat were dissociated enzymatically. The yield of
nodose neurons was suspended in Leibovitz L-15 medium supplemented with 10% (v/v) fetal bovine serum and
penicillin-streptomycin and plated onto No.1 glass coverslips.
Loading Asante Calcium Red indicator into neurons
Neurons were incubated for 50 min at room temperature with 3 μM Asante Calcium Red AM ester. Thereafter,
the cells were washed and maintained in fresh L-15 medium for 40 min to permit intracellular enzymatic
hydrolysis of the AM ester to proceed to completion.
Confocal fluorescence imaging
Indicator-loaded neurons were positioned on the stage of an inverted microscope (Axiovert 100 M, Zeiss) and
superfused with Locke solution (equilibrated with a mixture of 5% CO2 and 95% O2). Confocal imaging
microscopy was performed with the LSM 510 system (Zeiss) through a ⋅40 objective (N.A. 1.2; oil-immersion)
at a frame rate of 0.5 Hz. Excitation was at 488-nm; a 545-nm dichroic mirror separated the fluorescence
emission into two components: a short-wavelength component (designated F 525) that passed through a 500-550nm band-pass filter, and a long-wavelength component (F 650) that passed through a 560-nm longpass filter.
Neurons were stimulated with a 10-sec pulse of 100 μM ATP delivered through the superfusate.
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References
New product
Technical and scientific information
Related / associated products and documents
See BioSciences Innovations catalogue and e-search tool.
•
ASANTE Natrium™ Low Kd visible sodium
indicator, coming soon
•
Fura-2 AM, FP-42776C
Ordering information
Catalog size quantities and prices may be found at www.interchim.com/
Please inquire for higher quantities (availability, shipment conditions).
For any information, please ask : FluoProbes® / Interchim; Hotline : +33(0)4 70 03 73 06
Disclaimer : Materials from FluoProbes® are sold for research use only, and are not intended for food, drug, household, or cosmetic use.
FluoProbes® is not liable for any damage resulting from handling or contact with this product.
Asante is a trademark from Teflabs
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