Frequently Asked Questions

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Q & A on AcidiFluor™ ORANGE

No. Unfortunately AcidiFluor™ ORANGE cannot use for fixed samples. It is presumed that AcidiFluor ORANGE might not fluoresce in fixed samples because acidic environment does not maintain in acidic organelles by fixation. The acidic environment in acidic organelles seems to be realized using ATP as an energy source only when cells are alive.

AcidiFluor™ ORANGE is available for lysosomal imaging, the degranulation imaging of RBL-2H3 (shown in Application Note) and exocytosis imaging using neutrophil or insulinoma, etc. These applications are to be released in the Application Note as soon as we’re ready.

The fluorescence intensity spectra of AcidiFluor™ ORANGE measured in pH 3.0-pH8.0 buffer solutions are shown below. The smaller the pH value becomes, the intensity of fluorescence emission increases.

Using AcidiFluor™ ORANGE-NHS(GC302)  enables you to make the protein that emit fluorescence when it is taken into acidic organelles.

Intensity of AcidiFluor™ ORANGE doesn’t fluctuate very much in pH less than 3.

Q & A on AcidiFluor™ ORANGE-NHS

The labeling yield can be determined by using the following formula. Dividing the concentration of AcidiFluor™ ORANGE-NHS by the concentration of target protein, you can estimate the number of AcidiFluor™ ORANGE molecule binding to a target protein molecule.

A544, 280 : Absorbance of labeled protein at 544 nm or 280 nm

CF : Correction Factor (0.12)

e AcidiFluor ORANGE-NHS : Molar absorbance coefficient of AcidiFluor™ ORANGE-NHS (80,000)

e protein : Molar absorbance coefficient of target protein (ex. 216,000 for IgG)

We recommend the use of purified antibody or protein. If you want to label a mixture of proteins, please remove contaminating materials by ultrafiltration or gel filtration. Especially, contamination of amine (ex. Tris) causes the decrease of labeling efficiency.

Notably, if excess amount of dye binds to target protein (7-8 molecules per protein molecule), it incurs saturation of fluorescent signal or denaturation of target protein.

I’m sorry but AcidiFluor™ ORANGE-NHS is only for ‘live’ cell imaging. It is because acidic environment in the vesicles is ruined by fixing process.

Q & A on CaSiR-1™ / CaSiR-1™ AM

AM represents an acetoxymethyl group and AM ester is derivatized from carboxylic acid to add permeability to plasma membranes. As shown below, CaSiR-1™ AM permeates cell membrane and its acetoxymethyl group is cleaved by intracellular esterase to give CaSiR-1™ structure which can interact with Ca2+. Water solubility rises dramatically when acetoxymethyl group was cleaved to give CaSiR-1™, which become difficult to leak extracellular fluid.

CaSiR-1™ and CaSiR-1™ AM show little emission in the presence of up to 1mM Mg2+ or up to 100mM Na+ or K+. For details, please refer to the reference below.

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

Not only CaSiR-1™ AM but also all of AM ester probes are extremely high lipophilic. Therefore, in the case of AM esters are dissolved in DMSO and then dispersed in buffer solution directly, they aggregate and become difficult to be taken into the cell. So, it is necessary to prepare staining solution which is incorporated into cells well by adding detergent and in some cases, sonicate the solution of detergent and AM ester additionally.

Although we haven’t conducted cytotoxicity test yet, we are planning to compare cytotoxicity between our products and competitor goods. As soon as we experimented, we upload the results in HP. Also, since excitation light toxicity is small wave length > long wave length, near-infrared probe CaSiR-1 doesn’t harm cells very much compared to blue, green, or red Ca probe.


Because affinities are different.

POLARIC™ changes its colors by changing solvents, which we call solvato chromism. (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 ET(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 ET(30) value is small and long when ET(30) value is large.

The ET(30) value of each solvent

溶媒 Cyclohexane Toluene Dioxane THF Ethyl
CHCl3 CH2Cl2 Acetone DMF DMSO
ET(30)値 30.9 33.9 36 37.4 38.1 39.1 41.1 42.2 43.2 45.1


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

Intensity of light and color change simultaneously.

According to the difference of concentration

Though conventional probes only change intensity of light according to concentration etc., POLARIC™ probes not only change light intensity but also change colors simultaneously.

  1. As colors change, visibility improves dramatically.
  2. As colors change, quantitation increases.
  3. As colors change, it is possible to do multi color imaging which reflects on concentration distribution.

It is reported that the fluorescent wavelength of POLARIC™ is changed by the composition of lipid (Chem. Lett. 2011, 4, 989-991) , however, POLARIC™ has not significant difference of fluorescent wavelength by pH.

A safe and convenient tool to compose organic compounds.

Organic compounds are, of course, components of living things and used to make medicine, clothes, plastic and so on. As they are necessary to daily life, it is very important to “make organic compounds”.

When doing cooking, a variety of cooking tools are needed. In the same way, a variety of “tools (reactions)” are needed when composing organic compounds. This tool developed by Akira Suzuki (professor emeritus of Hokkaido University) plays a role of connecting objects and used all over the world to compose various organic compound because this is much safer and more convenient than existing methods.

This tool which generates organic compounds indispensable to our life brought significant benefits to human beings and awarded Nobel prize in 2010.

As the structures of POLARIC™ and lipids are resembled, hydrophilic part and hydrophobic part of POLARIC™ locate as lipids. Also, POLARIC™ emits fluorescence due to the environment where it locates by monodispersing.

Q & A on DAF, HPF, APF

NO immediately reacts with oxygen, and gives NO2 and NO3. DAFs react with intermediates, which are generated from NO and oxygen.  DAFs can detect NO specifically, because these intermediates cannot be produced without NO generation under physiological conditions.

DAF series will not react with NO2and NO3under physiological conditions. However, if they are incubated for a long time in the presence of high concentration NO2(≧10mM), slight fluorescence will be observed.

In the buffer of around pH 7 using DAF-2, NO detection limit is 5 nM. In case of using DAF-FM, it becomes slightly high sensitive as its intensity of fluorescence is 1.5 times higher than that of DAF-2

Pigmented substances such as phenol red and vitamins sometimes weaken the intensity of fluorescence. Proteins such as serum and BSA added to medium also sometimes lower the NO detection efficiency.

Fluorescence is observed by its accumulated signal because fluorescent compound produced by reaction of DAF reagent with NO is stable compound.  Therefore, when NO level decreases, fluorescence stops increasing but not decreasing. The slope of the curve of fluorescence intensity shows the production level of NO.

It is possible to determine absolute concentration of extracellular NO by using regents, DAF-2, DAF-FM, DAR-4M, without the cell membrane permeability. Please prepare calibration curve by the solution whose NO concentration is already known. It is difficult to determine NO concentration accurately for the reagents, DAF-2 DA, DAF-FM DA, DAR-4M AM, with the cell membrane permeability. This is because the reaction efficiency and quantum yield to detect NO will change due to the environmental difference in the cell.

For the determination of absolute concentration, it is necessary to use the NO gas solution of known density. However, practically, NONOate (NO of 2 molecules is generated by 1 molecule) is recommended to use.

The clear cytotoxicity was not recognized at the 10μM degree. In case the toxicity might be suspicious, please lower density.

Optimum concentration is about 5~10μM( diluted 500 ~ 1000 times). It may change slightly by the kind of sample and buffer used. Due to the properties of the fluorescein-based compound, it may have an adverse effect if the density of the reagent is raised for getting a strong signal.

It is difficult to measure the degradation in the cells directly, there is no data of direct measurement. Preliminary experiment using homogenates from brain tissue reveals that DAF-2DA was degraded within 10minites.

DAF-2 DA、DAF-FM DA are degradated by esterase and become DAF-2、DAF-FM. They leak outside cells gradually. Ater reacting with NO,  DAF-2T leak outside cells gradually.

Cultured cell, vascular endothelial cell, cranial nerve system such as hippocampal, peripheral blood mononuclear cell, earthworm ganglion, and plant cell are used for test sample. Image observation with the fluorescent microscope, measuring the fluorescence intensity of the specific points of the image, and measurement with multi-well plate reader measurement.

Yes, the application of DAF reagent measured by the flow site meter was reported by following papers.

  1. Havenga, M. J. E. et al. Simultaneous Detection of NOS-3 Protein Expression and Nitric Oxide Production Using a Flow Cytometer. Anal. Biochem. 2001, 290, 283-291.
  2. Navarro-Antolin, J. and Lamas, S. Nitrosative Stress by Cyclosporin A in the Endothelium: Studies with the NO-Sensitive Prove Diaminofluorescein-2/ diacetate using flow cytometry. Nephrol Dial Transplant, 2001, 16 [Suppl 1], 6-9.
  3. Jozsef L, Zouki C, Petasis NA, Serhan CN, Filep JG. Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-kappa B and AP-1 activation, and IL-8 gene expression in human leukocytes.  Proc Natl Acad Sci U S A 2002; 99: 13266-71.

No, you cannot detect in fixed cells/tissues.
Half-life of NO in physiological conditions is about one seconds. NO does not remain in fixed cells or tissues. Therefore, you cannot detect NO in fixed samples. Please use live cells or live/fresh tissues to detect NO using DAF/DAR series probes.

Yes. Excitation 495nm and fluorescence 515nm is the wavelength of efficiency peak.It can be measured if the wavelength is different slightly. (Anal. Chem. 1998, 70, 2450)It is reported that the measurement with long wave length have a the good result of sensibility and linearity in case there is an background with an admixture material.

Yes, it is. Of course bottom lead type is also possible.

There is no measurement data of DAF series incorporating into cells. However, For example, it takes 1 hour to load DAF-2 DA into aortal smooth muscle cell of rats. (Kojima, H. et al. Chem. Pharm. Bull, 1998, 46, 373-75) 30 minutes for DAR-4M AM loading into primary cultured endothelial cell. (Kojima, H. et al. Anal. Chem. 2001, 73, 1967-73) Please examine the optimal time by referring these reports.

APF have higher sensibility than that of HPF. HPF reacts with hydroxy radical and peroxynitrite. APF also reacts with hypochlorous ion.

APF and HPF have cell permeability? Therefore, they can be used for the intracellular imaging.

Please avoid freeze and thawing. 1mg of reagent is dissolved in 0.5ml DMSO in each package whose unit is indicated 1mg. Repeating freezing and thawing causes DMSO deterioration by water intakes. At the first time when you use the reagent, please divide it into small amount and store it. We recommend using up each pack when you use it. In order to avoid moisture absorption, when you take the vial out from the refrigerator, please leave the vial enough until the whole becomes the room temperature.

DAF and related products is not stable in aqueous medium. Please dilute the reagent just before use, and measure the fluorescence soon after the reaction. DAF is also unstable under strong light. Store the reagent at <-20℃, protecting from light.

Q & A on FeRhoNox-1

You can detect Fe(II) ion after fixation, if Fe(II) ion is not washed out. Or you can fix after staining of living cells with FeRhoNox-1. Please test the compatibility of FeRhoNox-1 with your cells and fixing protocol, because in some conditions, localization of Fe(II) might not be preserved, or fluorescence intensity might be decreased.

Q & A on NiSPY-3

This protocol is for the staining of HeLa cells. Add 2 μM NiSPY-3 to the culture medium (DMEM + 8% FBS + 100 units/mL penicillin + 100 mg/mL streptomycin) with 0.01% DMF and 0.01% Cremophor EL as cosolvent, and culture for 20 minutes. Then wash the cells with HBSS 2 times and observe by fluorescent microscopy. Use blue excitation filter set or 488 nm laser excitation. Post stimulation by peroxynitrite may increase the intracellular peroxynitrite concentration and increase in the fluorescence of NiSPY-3.

Q & A on MAR

From a vial of MAR (25 μg) , ~43 μL of 1 mM stock solution can be made. If you use 1 mL of 1 μM MAR solution for one assay, you can perform ~40 assays per one vial.

MAR, a material with no fluorescence fluoresces in hypoxic environment. Because this reaction is irreversible, you can dissociate cells by trypsin and dilute cells into normal buffers or medium without decreasing the MAR’s fluorescence.

Yes, mild fixation does not bleach the fluorescence, however, strong fixation condition may alter the intracellular localization of MAR or decrease the fluorescence. Please test the fixation condition in your cells and conditions.

Q & A on KP-1

We did not observe cytotoxic effects of KP-1 in 1μM, a concentration typically used. We tested 48-hour culture in the presence of 1 μM KP-1 and found it to not affect the cell viability.

Q & A on GlycoYELLOW-βGal

You may use a similar procedure to X-gal staining for fixed cell samples, because GlycoYELLOW-βGal is also a substrate for β-galactosidase (lacZ reporter)

We confirmed that fluorescence of GlycoYELLOW-βGal observed in live cells does not diminish by the normal process of fixation. Please refer the following Application Notes for details.

Strong fixation conditions may alter the intracellular localization or may decrease the fluorescence. Please test the fixation condition in your cells and conditions.

Yes, using GlycoYELLOW-βGal, you can detect enhancement of β-galactosidase activity caused by senescence (SA-βGal).

If physiological β-Gal activity in lysosomes is detected with GlycoYELLOW-βGal, then you may use bafilomycin A1 for alkalization of lysosomes and inhibit physiological β-Gal activity. You may not need to use alkalization reagent if you do not detect physiological β-Gal.

Q & A on Fluorescence observation

It is unaffected in most cases. Especially in case of ROS detection probes, ROS increases under starvation for certain types of cells. It would be better to add the fluorescence probe into the culture medium, not the buffer such as HBSS. However, components (such as phenol red which is used as an indicator) in the medium might influence the detection. Therefore, please follow the information in an instruction manual or literature.

It is not always necessary to replace the solution before observation, in case of high-efficiency fluorescence probes which do not emit fluorescence light before reacting with target substances. The signal could be detected even when the unreacted probes remain in the culture medium. However, it might be necessary to replace the solution to remove fluorescence substances, especially when the medium containing phenol red interferes optically with the fluorescent observation. (Please refer the below illustration.) In this case, by using the medium which does not contain phenol red, it is able to observe without the solution replacement. It is recommended to determine the solution condition, based on preliminary experiments with the target cells and the medium

The fluorescence spectra of DMEM medium when exciting at different wavelengths (slit width 20 nm). Fluorescence emission from green to red were observed when exciting with the blue light.

Usually, glass bottom dish is made for fluorescence microscopy observation. Use glass bottom dish that fit the stage of your microscope. In some cases, you may use multi-well plates instead of glass-bottom dish.

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