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[Near-Infrared Ca2+ probe] CaSiR-1™ / CaSiR-1™AM

  • Fluorescence at near-infrared area
  • Large Increase in Fluorescence Intensity upon Binding Ca2+
  • Usable for Live Cell Imaging

CaSiR-1™ and CaSiR-1™ AM are near-infrared fluorescence calcium probes which have fluorescence maximum wavelength at 664 nm. Multicolor imaging is possible between CaSiR-1™ or CaSiR-1™ AM and fluorescent probes or fluorescent proteins which have fluorescent wavelength in visible area such as Hoechst, Fluorescein, Rhodamine, GFP, YFP and RFP etc. Near-infrared region has greater tissue penetration, less overlap with the spectrum of background autofluorescence and exhibits less phototoxicity to cells and tissue.

CaSiR-1™ changes fluorescent intensity greatly when it binds to calcium. For example, fluorescent intensity rises more than 1000-fold when calcium concentration is changed from 0 µM to 39 µM. Little fluorescence is detected when calcium concentration is 0 µM.

CaSiR-1™ is suited to cell introduce by microinjection, patchclamp and electroporation, etc. CaSiR-1™ AM, an acetoxymethyl ester of CaSiR-1™, can permeate cell membrane. After permeating cell membrane, CaSiR-1™ is hydrolyzed by esterase to give CaSiR-1™ and stay in the cell. From these methods, it is possible that catching fluctuation of intracellular calcium concentration as the change of fluorescence intensity in CaSiR-1™ induced living cells. Action potential can be regarded as calcium concentration fluctuation in the cell when CaSiR-1™/CaSiR-1™ AM is used to neuron.

Price
Code No. Product Size Price Protocol MSDS
GC401 CaSiR-1™ 1 mg USD $ 780.00
  • Donwload
    552KB
  • Donwload
    161KB
GC402 CaSiR-1™ 50μg×20 $890.00
  • Donwload
    199KB
GC403 CaSiR-1™ AM 50μg×20 $990.00
  • Donwload
    200KB

The feature of CaSiR-1™ AM / CaSiR-1™

Kd for Ca2+ 0.58 μM

Excitation Wavelength 650 nm

Fluorescent Wavelength 664 nm

Quantum Yield 0.20

1. Fluorescence at near-infrared area

Ca2+ imaging of mouse cranial nerve
Fig.1 Ca2+ imaging of mouse cranial nerve

CaSiR-1™ AM was loaded to a mouse brain slice in which a fraction of neurons expressed Venus (a mutant of YFP) and Ca2+ imaging was conducted. CaSiR-1™ is shown as red color and Venus is shown green. Fluorescent signal is change visualized a transient elevation of intracellular Ca2+ concentration associated with Ca2+ spark of cranial nerves. Thus, this experiment was conducted to compare Ca2+ behavior at neurons when YFP is expressed and not expressed. Various multicolor imaging are possible, including Ca2+ imaging of the samples which specific cells are labeled by fluorescent protein.

2. Large Increase in Fluorescence Intensity upon Binding Ca2+

Fluorescent spectra of CaSiR-1™
Fig.2 Fluorescent spectra of CaSiR-1™

In the presence of various concentration of Ca2+ (0, 0.017, 0.038, 0.065, 0.100, 0.150, 0.225, 0.351, 0.602, 1.35, 39µM), fluorescent spectra of CaSiR-1™ was measured in the 30mM of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH7.2) including 100 mM KCl and 10 mM ethylene glycol tetraacetic acid (EGTA). The excitation wavelength was 620nm. When Ca2+ concentration was fluctuated from 0 µM to 39 µM, fluorescent intensity raised more than 1000-fold.

3. Usable for Live Cell Imaging

Ca2+ imaging of HeLa cell
Fig.3 Ca2+ imaging of HeLa cell

CaSiR-1™ was loaded to HeLa cell and stimulated by ATP and ionomycin. Although a part of probe localized to lysosome, it was possible enough to visualize Ca2+ concentration change.

Protocol of the experiment above; staining of HeLa cells.
  1. 2 mM dimethylsulphoxide (DMSO) solution of CaSiR-1™ AM were mixed with an equal volume of 20% (w/v) Pluronic F-127 in DMSO to receive 1 mM stock solution.
  2. Aliquots were diluted with HBSS buffer to give final concentrations of 3µM as a staining solution. Final concentrations of Pluronic F-127 is 0.03%.
  3. Culture medium were removed from glass bottom dish and HeLa cells were washed with HBSS 3 times.
  4. Cells were loaded with the staining solution for 30 minutes, under 37℃, 5 % CO2.
  5. Staining solution was removed, the cells were washed with HBSS 3 times and replaced by HBSS. And then the changes of intracellular fluorescence intensity were observed in response to agonists.
Reference
F. Zhang, E. S. Tzanakakis (2017)
Scientific Reports 7:9357, DOI: 10.1038/s41598-017-09937-0
Egawa T., Hirabayashi K., Koide Y., Kobayashi C., Takahashi N., Mineno T., Terai T., Ueno T., Komatsu T., Ikegaya Y., Matsuki N., Nagano T., Hanaoka K. Angew. Chem. Int. Ed. 2013, 52, 3874 -3877.
Egawa, T.; Hanaoka, K.; Koide, Y.; Ujita, S.; Takahashi, N.; Ikegaya, Y.; Matsuki, N.; Terai, T.; Ueno, T.; Komatsu, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 14157-14159.
Mika Mizunuma, Hiroaki Norimoto, Kentaro Tao, Takahiro Egawa, Kenjiro Hanaoka, Tetsuya Sakaguchi, Hiroyuki Hioki, Takeshi Kaneko, Shun Yamaguchi, Tetsuo Nagano, Norio Matsuki and Yuji Ikegaya, Nat. Neurosci., 17, 503-505 (2014), “Unbalanced excitability underlies offline reactivation of behaviorally activated neurons”

CaSiR-1™ and CaSiR-1™ AM were commercialized by Goryo Chemical Company under the guidance of Prof. Tetsuo Nagano (Professor of Laboratory of Chemistry and Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo).

Contact Information

Further information or questions on our products, please contact us, E-mail: info_itnl@goryochemical.com