POLARIC™ changes its colors by changing solvents, which we call solvatochromism. (left side picture indicates.)

Solvents have an index called polarity. Affinity of POLARIC™ fluorescent probes for solvents is influenced by solvent polarity, such as water-like or oil-like. This changes dissolved POLARIC™ fluorescent probes, for example, blue for salad oil, green for alcohol and red for water. As a result, difference of affinity between POLARIC™ fluorescent probes and solvents can be showed as variation of colors.

This character allows us to replace various phenomenon such as temperature change, pressure change, pH change and hardness change etc. into color change of POLARIC™ fluorescent probes.

FIG.1 Pattern diagram of Jabronski Diagram and solvent reorientation

After POLARIC™ fluorescent probes are excited by light absorption, their excitation state are stabilized by reorientation of surrounding solvents.
Since electronic charge of probe molecules in excitation state are largely localized, polar solvent is more efficient to stabilize excitation state than non-polar solvent. The difference of stabilization reflects on the difference of fluorescence wavelength.

The E_T(30) value of each solvent is shown in Table. As FIG.2 shows, when POLARIC™ are solved in each solvent, the color changes from blue (left side) to yellow (right side). This is because the wavelength is short when E_T(30) value is small and long when E_T(30) value is large.

FIG.2 UV image photograph of each solvent which dissolved POLARIC™

Summery

CaTM-2™ / CaTM-2™ AM are red-colored fluorescent calcium probes including TokyoMagenta, a new red-colored fluorescent probe. They are suited to analyze calcium ion behavior in cytoplasm very well and emit fluorescent with high sensitivity due to Ca²⁺ concentration.

Multicolor imaging is possible between CaTM-2™ or CaTM-2™ AM and Hoechst, Fluorescein, Rhodamine, GFP, YFP and RFP etc., which are fluorescent probes or fluorescent proteins having fluorescent wavelength from UV region to visible area. The advantages of long wavelength region are greater tissue penetration and low phototoxicity.

Dissociation constant (Kd) between CaTM-2™ / CaTM-2™ AM and Ca²⁺ is 0.20µM. CaTM-2™ / CaTM-2™ emit red fluorescence due to concentration of Ca²⁺ sensitively.
CaTM-2™ / CaTM-2™ AM are calcium probes which distribute uniformly in cytoplasm when loaded to cell and well-suited to analyze Ca²⁺ concentration at cytoplasm.

The feature of CaTM-2™ AM / CaTM-2™

• Fluoresce at red region
• Recognize Ca²⁺ supersensitivity.
• Visualize fluctuation of Ca²⁺ in cytoplasm

Spontaneous combustion of nerve cells at a slice of rats brain.

（a－c）Image of CaTM-2 AM（a）, fluorescent image of acridine orange which was used for fixing cells（b）and two fluorescent images on top of one another (c）. （d）Time change of CaTM-2 fluorescent intensity. Fluctuation of Ca²⁺ concentration in the cell along with combustion of nerve cells is captured as fluorescent intensity at places of ～ in (a)

Live cell staining protocol with CaTM-2™ AM

Materials Required but not Provided

• Anhydrous dimethylsulfoxide (DMSO)
• Appropreate medium or buffer such as Hank’s Balanced Salt Solution (HBSS)
• 20 % Pluronic F-127 in DMSO

Preparation of Reagent and Cell Staining

1. To prepare a stock solution, dissolve the CaTM-2™ AM 50µg in 41 µL of DMSO to 1 mM. To improve the induction efficiency and inhibit localization of the probe, the addition of Pluronic F-127 is recommended.
2. Dilute an aliquot of stock solution to a final concentration of 1-10 µM in appropreate loading medium or buffer such as HBSS(stain solution). Final concentration of Pluronic F-127 is around 0.01-0.05 %.
3. Remove the culture medium from cell culture dish and wash with loading medium.
   Caution : Glass bottom dish etc. are recommended as a cell culture dish, because it has no intrinsic fluorescence.
4. Add stain solution to the dish and incubate 10 - 60 minutes under 37 ℃, 5 % CO2 conditions.
5. After staining, remove the stain solution from the dish and wash 2 or 3 times by medium or buffer which is not contained probe. Replace to HBSS buffer and observe the changes of intracellular fluorescence intensity using a fluorescence microscopy.

Observation Fluorescence

597 nm is suited as excitation wave length. Texas Red、Y-2E/C（Nikon Inc.）or U-FYW,U-MWIY2（Olympus Inc.）etc. can be used as a filter.. Maximum fluorescent intensity is detected at 609 nm.

Storage

Probes are shipped in the conditions of nitrogen-shield and dry. After receiving, please storage at dry and cool space (under -20 ℃. After resolving in DMSO, we recommend to use up at once.

Reference

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.

CaTM-2™ and CaTM-2™ were commercialized by Goryo Chemical Company under the guidance of prof. Tetsuo Nagano (Graduate School of Pharmaceutical Sciences, Laboratory of Chemistry and Biology). These products were developed with support from JST program “Development of System and Technology for Advanced Measurement and Analysis”.

Inquiry for products and services

If you are interested in our services and products in this page or want to purchase them, please contact us,

E-mail: solvatopolaris-t.com

• 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.

The feature of CaSiR-1™ AM / CaSiR-1™

1. Fluorescence at near-infrared area

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⁺

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

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.

 

General protocol is below

• Download protocol （PDF 552KB）

Reference

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

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

Contact Information

Further information or questions on our products, please contact us, E-mail: solvatopolaris-t.com

• Fluoresce at red region
• High sensitivity to Ca²⁺
• Visualize fluctuation of Ca²⁺ in cytoplasm

CaTM-2™ / CaTM-2™ AM are red fluorescent calcium probes based on TokyoMagenta, a new red-colored fluorophore. They are suited to analyze calcium ion behavior in cytoplasm very well and emit fluorescence with high sensitivity due to Ca²⁺ concentration.

CaTM™-2 and CaTM™-2 AM are red-colored fluorescent calcium probes which have maximum fluorescence wavelength at 609 nm. Multicolor imaging is possible between CaTM-2™ or CaTM-2™ AM and Hoechst, Fluorescein, Rhodamine, GFP, YFP and RFP etc., which are fluorescent probes or fluorescent proteins having fluorescent wavelength from UV region to visible area. The advantages of long wavelength region are greater tissue penetration and low phototoxicity.

Dissociation constant (K_d) between CaTM-2™ / CaTM-2™ AM and Ca²⁺ is 0.20 µM. CaTM-2™ / CaTM-2™ AM emit red fluorescence due to concentration of Ca²⁺ sensitively.

CaTM-2™ / CaTM-2™ AM are calcium probes which distribute uniformly in cytoplasm when loaded to cell and well-suited to analyze Ca²⁺ concentration at cytoplasm.
CaTM-2™ is suited to cell introduce by microinjection, patchclamp and electroporation, etc. CaTM-2™ AM, an acetoxymethylester of CaTM-2™, can permeate cell membrane. After permeating cell membrane, CaTM-2™ is hydrolyzed by esterase to give CaTM-2™ and stay in the cell. From these methods, it is possible that catching fluctuation of calcium concentration in the cell as the change of fluorescent intensity in CaTM-2™ induced living cells.

The feature of CaSiR-2™ AM / CaSiR-2™

1. Fluoresce at red region

Fig.1 Multicolor imaging using HeLa cells

HeLa cell, expressed CFP at nucleus and YFP at golgi, was introduced CaTM™-2 AM to do multicolor imaging. CaTM™-2 AM can be available for various multicolor imaging such as expressing fluorescent protein to organelle and doing calcium imaging at labeled cells.

2. Recognize Ca²⁺ supersensitivity.

The status of CaTM-2™ in the presence of Ca²⁺

When CaTM™-2 recognizes calcium ion, fluorescent intensity rises dramatically.

Fig.2 The fluorescence feature of CaTM-2™

This graph shows spectral change accompanied to fluctuation of Ca²⁺ concentration in the solution which includes CaTM-2™. An increase of red fluorescent (609 nm) derived from fluorescent probe is observed.

3. Visualize fluctuation of Ca²⁺ in cytoplasm

Fig.3 Imaging of intracellular Ca²⁺ concentration fluctuation by histamine stimulation.

(a - c) Fluorescent image of HeLa cell which CaTM™-2 AM was introduced and was stimulated by Histamine (a, b). Temporal change of fluorescent intensity at each cell shown in (a) by 1～3 (c). It is observed CaTM-2 distributes in cytoplasm and catches the fluctuation of Ca2+ concentration.
(d-f) By use of traditional red-colored calcium probe (Rhod-2 AM), the same experiment as (a‐c) was conducted. As Rhod-2 has a tendency to localize in mitochondria, only Ca²⁺ concentration in mitochondria can be observed.

Reference

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.

CaTM-2™ and CaTM-2™ were commercialized by Goryo Chemical Company under the guidance of Prof. Tetsuo Nagano. (Graduate School of Pharmaceutical Sciences, Laboratory of Chemistry and Biology) These products were developed with support from JST program “Development of System and Technology for Advanced Measurement and Analysis”.

Contact us

If you are interested in our services and products in this page or want to purchase them, please contact us, E-mail: solvatopolaris-t.com