Ca Chelators

Appearance: white powder or crystalline powder
Purity: >95.0 % (Tit.)
Water content: 3.5-14.0 %
Sulfated ash: 46-53 %
Solubility: 10 mg/ml water
pH: 8.0-9.5

BAPTA is a calcium-selective chelator developed by Dr. Tsien. It has log_KCa=6.97 and log_KMg=1.77. The basic chelating unit resembles that of EGTA, but the two aliphatic nitrogen atoms are replaced by aromatic nitrogen. Thus, BAPTA is not protonated at physiological pH. BAPTA possesses pKa₃=5.47 and pKa₄=6.36. This property indicates that the deprotonation step is not included in its calcium complexation step, and it has a higher complexation rate than EGTA because it is not affected by proton interference. BAPTA-AM is an acetoxymethyl ester derivative of BAPTA that can be easily loaded into cells using the AM method. BAPTA-AM is useful for controlling the intracellular calcium concentration.

Appearance: white powder or crystalline powder
Purity: >95.0 % (Tit.)
Water content: 3.5-14.0 %
Sulfated ash: 46-53 %
pH: 8.0-9.5

Appearance: white crystalline powder
Purity: >98.0 % (Tit.)
Sulfated ash: <0.1 %
Heavy metal (as Pb): <0.001 %
Fe content: <0.001 %
Solubility: 1 g/10 ml 1 M NaOH -> 100 ml water

EGTA is the most widely used calcium selective chelator. The calcium complex of EGTA is 100,000 times more stable than its Mg complex. It is utilized to prepare calcium buffers and control the calcium ion concentration.

Product Name: Fluo 3

Product Code: F019-10Unit: 1 mg

Description:

Chemical Name: 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid

Appearance: red solid
Fluorescent spectrum: pass test
Solubility: 2 mg/ml acetonitrile, 0.9 mg/ml DMSO
Licensed under U.S. Pat. Appl. No. 4,603,209

Fluo 3 is a long wavelength calcium probe. Fluo 3 is practically non-fluorescent in its free ligand form, but its fluorescence increases 60-80 times when it forms complexes with calcium. Thus, it has been widely used with confocal laser fluorescent microscopy because the microscope has an argon laser. The long wavelength of the fluorescent signal is also convenient for minimizing photodamage to sample cells. Fluo 3 is also useful for caged calcium and others that are cleaved by the photo-irradiation in the UV region. Fluo 3-AM is an acetoxymethyl ester derivative of Fluo 3 that can be easily loaded into cells by incubation.

General Protocol (for Human T cells)*
Reagents:
2 mM Fluo 3-AM/DMSO (1 mg Fluo 3-AM in 442 ml DMSO)
Pluronic F127
Hanks·balanced salt solution (HBSS)
HEPES buffer saline (10 mM HEPES, 1 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, 0.1% BSA, pH 7.4)
Protocol:
1. Add 16.5 mg Pluronic F127 to Fluo 3-AM/DMSO solution. Pluronic F127 prevents aggregation of Fluo 3-AM in HBSS and helps uptake with cells.
2. Dilute the Fluo 3-AM solution with HBSS to prepare 4 mM Fluo 3-AM working solution.
3. Add the Fluo 3-AM working solution to the cells, and incubate at 37 oC for 20 min.
4. Add 5 volumes of HBSS containing 1% fetal calf serum, and continue the incubation for another 40 min.
5. Wash the cells 3 times with HEPES buffer saline. Then resuspend the cells to prepare 1x105 cells/ml solution using HEPES buffer saline.
6. Incubate at 37 oC for 10 min. Then use the cells for fluorescent calcium ion detection.
7. Monitor the fluorescence at 528 nm (excitation: 490-500 nm).
*Cell staining conditions differ by cell types, so it is necessary to optimize the conditions for each experiment.

Product Name: Fluo 3-AM

Product Code: F023-10 Unit: 1 mg

Description:

Chemical Name: 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5- methylphenoxy) ethane-N,N,N',N' - tetraacetic acid, pentaacetoxymethyl ester

Appearance: red solid
Fluorescent spectrum: pass test
Solubility: 2 mg/ml acetonitrile, 0.9 mg/ml DMSO
Licensed under U.S. Pat. Appl. No. 4,603,209

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Product Name: Fura 2

Product Code: F014-10 Unit: 1 mg

Description:

Chemical Name: 1-[6-Amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N',N' -tetraacetic acid, pentapotassium salt

Appearance: yellow or yellowish orange solid
Purity: >98.0 % (HPLC)
Fluorescent spectrum: pass test
lex: 380 nm, lem: 510 nm (free)
lex: 340 nm, lem: 510 nm (Ca complex)
Solubility: 4 mg/ml water
Licensed under U.S. Pat. Appl. No. 4,603,209

Fura 2 was developed to improve the fluorescent properties of Quin 2. The signal intensity in 1 mM of loaded Fura 2 corresponds to that of 30 mM of loaded Quin 2. This allows an experiment at a lower concentration of indicator using Fura 2 as compared to Quin 2. Fura 2 is one of the most widely used calcium indicators for ratiometric measurement. Many types of instrumentation are now available for experiments using Fura 2. Fura 2 is especially suitable for digital imaging microscopy. It is less susceptible to photobleaching than Indo 1. Changes in the cell shape can sometimes affect the fluorescent ratio at 340 nm and 380 nm. For example, fluorescent signal intensities at these wavelengths sometimes decrease simultaneously with smooth muscle contraction. For blood vessels, however, the increase of the signal intensity at 340 nm tends to be smaller on contraction, while the decrease of the signal intensity at 380 nm tends to be larger with its contraction. Fura 2-AM is an acetoxymethyl ester derivative of Fura 2 that can be easily loaded into cells by incubation.

General Protocol (for NG 108-15/ Neuronal Cell Line)*
Reagents:
1 mM Fura 2-AM/DMSO (1 mg Fura 2-AM in 1 ml DMSO)
Hanks·balanced salt solution (HBSS)
HEPES buffer saline (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 13.8 mM glucose, pH 7.4)
Protocol:
1. Culture cells on a glass-bottom dish using DMEM containing 5% fetal calf serum.
2. Change the medium to 1 mM dibutyl cAMP/DMEM, and culture the cells for 3-4 days to induce dendrites.
3. Dilute 1 mM Fura 2-AM DMSO solution with HEPES buffer saline to prepare 1 mM Fura 2-AM working solution.
4. Remove the culture medium, and add 0.5 ml of the Fura 2-AM working solution to the cells.
5. Incubate for 20 min. Then remove the Fura 2-AM working solution.
6. Wash the cells once with HEPES buffer saline. Then incubate the cells for 1 hour in the HEPES buffer saline.
7. Use the cells for fluorescent calcium ion detection.
8. Monitor the excitation spectra at 380 nm (calcium free) and 340 nm (calcium complex) with fixed emission at 510 nm.
*Cell staining conditions differ by cell types, so it is necessary to optimize the conditions for each experiment.

Product Name: Fura 2-AM

Product Code: F015-10 Unit: 1 mg

Description:

Chemical Name:-[6-Amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester

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Product Name: Indo 1

Product Code: I004-10

Unit: 1 mg

Price (USA and Canada): $85

Availability: ships in 5-10 days

Description:

Storage: -20 ºC, protect from light

Shipping Condition: Ambient Temperature

Chemical Name: 1-[2-Amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid, pentapotassium salt

CAS Number: 132319-56-3, 96314-96-4, free

Appearance: slightly yellowish off white powder
Purity: > 95% (HPLC)
Fluorescent spectrum: pass test
lex: 330 nm, lem: 485 nm (free)
lex: 330 nm, lem: 410 nm (Ca complex)
Solubility: 4 mg/ml water
Licensed under U.S. Pat. No.4,603,209

Indo 1 is another type of improved calcium indicator that can be used in ratiometry. Two separate wavelengths in the emission spectrum, usually at 410 nm and 480 nm, are measured in the ratiometry of Indo 1. Indo 1 is suitable for ratiometry using a flow cytometer that can measure fluorescent signals at dual wavelengths. Indo 1 is reported to have better properties for localization than Fura 2 after being loaded into cells. Indo 1-AM is an acetoxymethyl ester derivative of Indo 1 that can be loaded into cells by incubation.

Product Name: Indo 1-AM

Product Code: I005-10 Unit: 1 mg

Description:

Chemical Name: 1-[2-Amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, pentaacetoxymethyl ester

Appearance: yellowish off white powder
Purity: >97.0 % (HPLC)
Fluorescent spectrum: pass test
lex: 330 nm, lem: 485 nm (free)
lex: 330 nm, lem: 410 nm (Ca complex)
Solubility: 4 mg/ml water
Licensed under U.S. Pat. No.4,603,209

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Product Name: Quin 2

Product Code: Q001-11 Unit: 100 mg

Description:

Chemical Name: 8-Amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid, tetrapotassium salt

Appearance: pale yellow powder
Purity: >95.0 % (HPLC)
Molar absorptivity: >37,000 (240 nm)
Water content: 11-13 %
Solubility test: 0.8 mg/ml water

Quin 2 forms a stable fluorescent complex with calcium (logK_CaY=7.1), but not with magnesium (log_MgY=2.7). The complex has a high quantum yield (0.14) at emission wavelength 525 nm, excitation wavelength 339 nm, and emission wavelength 492 nm. Quin 2-AM is an acetoxymethyl ester derivative of Quin 2 that is readily permeable through cell membranes. Within the cell, the ester is hydrolyzed to Quin 2.

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Product Name: Rhod 2

Product Code: R001-10 Unit: 1 mg

Description:

Chemical Name:1-[2-Amino-5-(3-dimethylamino-6-dimethylammonio-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, chloride

Appearance: dark purple solid
Purity: >60.0 % (HPLC)
lex: 533 nm, lem: 576 nm (Ca complex)
Solubility: 50 mg/ml 0.1 M NaOH
Licensed under U.S. Pat. Appl. No. 115,921

Rhod 2 has the longest fluorescent wavelength signal of all the calcium indicators. It has a rhodamine-like fluorophore whose excitation and emission maxima are 557 nm and 581 nm, respectively. This makes it a convenient excitation source for argon and krypton lasers. Although it was thought that the fluorescent signal of Rhod 2 only increases several times with the calcium complex, Dojindo? Rhod 2 increases its signal with calcium 80-100 times because of its high purity. Its signal intensity is the strongest of all the calcium probes. Rhod 2 is thus highly recommended as a probe for intracellular calcium monitoring using laser microscopes. Rhod 2 is reported to have a better loading profile at the point of localization, especially in neural slice cultures. The dissociation constant of Rhod 2 with calcium (K_d=1.0 mM) is the highest of all the fluorescent calcium probes, providing a wider range for monitoring calcium concentration. Rhod 2-AM is an acetoxymethyl ester derivative of Rhod 2 that can be easily loaded into cells using the AM method.

Product Name: Rhod 2-AM

Product Code: R002-10 Unit: 1 mg

Description:

Chemical Name:1-[2-Amino-5-(3-dimethylamino-6-dimethylammonio-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester, chloride

Appearance: dark purple solid
Purity: >60.0 % (HPLC)
lex: 533 nm, lem: 576 nm (Ca complex)
Solubility: 50 mg/ml 0.1 M NaOH
Licensed under U.S. Pat. Appl. No. 115,921

Appearance: yellow powder
Purity: >95.0 % (HPLC)
lex: 355 nm, lem: 460 nm
Solubility: 3 mg/ml 50 % acetonitrile/water

MQAE with bromide ion as a counteranion has fluorescence excitation at 355 nm and emission at 460 nm. The fluorescent intensity of MQAE decreases in proportion to the chloride increase in cells. Chloride ion concentrations less than 50 mM may be monitored without being affected by pH changes. MQAE has high water solubility and membrane permeability.

General Protocol*

Reagents:
5 mM MQAE/Krebs-HEPES buffer
Krebs-HEPES buffer (20 mM HEPES, 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl₂, 1 mM MgCl₂, 16 mM glucose, pH 7.4)

Protocol:
1. Wash the cells with Krebs-HEPES buffer 3 times.
2. Add 5 mM Cl probe solution to the cells, and incubate at 37 ºC for 1 hour.
3. Wash the cells with Krebs-HEPES buffer 5 times.
4. Determine the fluorescence intensity using a fluorescence microscope coupled with an image analyzer.
* Cell staining conditions differ by cell type, so it is necessary to optimize the conditions for each experiment

Appearance: white or slightly yellow crystals
Purity: >98.0 % (Tit.) m.p.: 110-115 ºC
Solubility: 10 mg/ml DMSO, 0.6 mg/ml acetic acid

TPEN is a highly selective chelator of transition metal cations because the pyridine groups act as soft electron donators. TPEN is thus utilized for masking transition metal cations in fluorescent calcium monitoring. The masking function of the zinc cation is important for canceling the fluorescent artifact. TPEN is water-soluble and cell membrane permeable. Maximum wavelength is 260 nm.The physical properties of TPEN are as follows: pKa₁=10.27, pKa₂=3.32, pKa₃=4.85, and pKa₄=7.19; LogK_Mn=10.27, logK_Fe=14.61, logK_Zn=15.58, logK_Mg=1.7, and logK_Ca=4.4.

Appearance: colorless pellet

Inositol phosphates and diacylglycerols are generated from phosphatidyl inositols by phospholipase C. They are involved in cellular calcium ion movements as second messengers. Inositol-1,4,5-triphosphate (Ins(1,4,5)P₃) is an extremely important messenger that triggers calcium ion release from endoplasmic and sarcoplasmic reticulum. It is thought to increase the calcium sensitivity of the inositol induced calcium release (IICR) channel in the intracellular calcium source. Thus, the IICR channel can be opened with a low level of calcium ion concentration, causing a dramatic calcium burst into the cytosol. The released calcium ions and diacylglycerol then activate protein kinase C, which is one of the key enzymes in signal transduction.

Appearance: reddish-brown or red crystalline powder
Purity: >90.0 % (HPLC)
Fluorescent spectrum: pass test
Solubility: 2 mg/ml acetonitrile, 0.8 mg/ml DMSO
Packaged in nitrogen gas

BCECF is the most widely used intracellular pH probe. Dr. Tsien and others improved this carboxyfluorescein by introducing two extra carboxylates that allow it to be retained better by the cell. BCECF is highly water-soluble because it has 4 to 5 negative charges at neutral pH; it becomes difficult to pass through the cell membrane after loading. Its pKa value, 6.97, is higher than that of carboxyfluorescein. BCECF has an isosbestic point at 439 nm in the excitation spectra, so it can be used in ratiometry, similar to Fura 2. Wavelengths of 505 nm and 439 nm are usually used for the ratiometric assay, and 490 nm and 450 nm filters are set in front of the excitation light source. The 530 nm filter is used for its fluorescent signal. Please note that the excitation spectrum is slightly different from the absorption spectra. BCECF-AM is an acetoxymethyl ester of BCECF. It enables the easy loading of BCECF into cells. BCECF-AM accumulates in a cell only by incubation as do the other acetoxymethyl esters. BCECF-AM is very sensitive to moisture; it should be carefully handled. The color of the DMSO solution changes from pale yellow to dark orange with decomposition of the AM form. Therefore, hydrolysis of the AM ester can be monitored by changes in color.

General Protocol (for Human Neutrophil)*

Reagents:
1 mM BCECF-AM/DMSO solution (1 mg BCECF in 1.45 ml DMSO)
HEPES buffer saline (20 mM HEPES, 153 mM NaCl, 5 mM KCl, 5 mM glucose, pH 7.4)

Protocol:
1. Suspend cells in HEPES buffer solution to prepare 4x107 cells/ml.
2. Add 1 mM BCECF-AM/DMSO solution to the cell suspension to prepare 3 mM BCECF-AM (1/300 vol of cell suspension) as the final concentration.
3. Incubate the cell suspension at 37 oC for 30 min.
4. Wash the cells 3 times with HEPES buffer saline, and then prepare 3x106 cells/ml of the cell suspension.
5. Determine the fluorescence intensity using a fluorescence microscope or a confocal laser microscope coupled with an image analyzer.
* Cell staining conditions differ by cell type, so it is necessary to optimize the conditions for each experiment.

Appearance: orange or orange-brown crystals
Purity: >90.0 % (HPLC)
Fluorescent spectrum: pass test
Solubility: 2 mg/ml acetonitrile, 0.8 mg/ml DMSO
Packaged in nitrogen gas

Appearance: slightly yellow crystalline powder
Purity: >95.0 % (HPLC)
lex: 323 nm, lem: 528 nm (Zn complex)
Solubility: 1 mg/ml water

Dansylaminoethyl-cyclen is a water-soluble and cell membrane permeable fluorescent zinc indicator developed by Dr. E. Kimura. The aqueous solution of this reagent allows intracellular zinc ion to be monitored. The fluorescence intensity of the complex is 5 times that of the free ligand. Most other cations do not form fluorescent complexes with this reagent. Although cadmium ion forms a fluorescent complex, the stability constant of this complex is 10 times less than that of the zinc complex. Thus, cadmium ion does not affect zinc detection in most cases. Copper ion (Cu(II)), which forms a non-fluorescent complex, also does not affect zinc detection because it is extremely rare as a free ion in normal living cells. Recently, the zinc ion has gained a lot of attention; it is thought to play an important role in ischemia and subsequent selective neuronal cell death. Reactive oxygen species might damage cystein-residues in a zinc-finger motif to produce free zinc ions.

Appearance: white or slightly blue crystalline powder
Purity: >98.0 % (HPLC)
Fluorescent blank: pass test
lex: 368 nm, lem: 490 nm (Zn complex)
Solubility: 2 mg/ml DMSO

Zinquin ethyl ester is an analog of the widely used indicator TSQ. Though Zinquin ethyl ester itself is fluorescent, its fluorescence intensity is negligibly weak (1/30). Zinquin ethyl ester is membrane permeable, as are acetoxymethyl esters of calcium probes such as Fura 2-AM and Fluo 3-AM. Zinquin ethyl ester is thus useful to detect intracellular zinc ions. It forms a complex with a zinc ion with nitrogen atoms in the structure. This compound also forms a fluorescent complex with cadmium ion; however, detectable amounts of cadmium ions are not contained in normal living cells. Because the water solubility of Zinquin ethyl ester is poor, dimethylsulfoxide (DMSO) or ethanol is required as a solvent for preparing the stock solution.

General Protocol for Lymphoblastoid Cells*

Reagents:
2.4 mM Zinquin ethyl ester stock solution (1 mg Zinquin ethyl ester in 1 ml DMSO)
Zinquin ethyl ester stock solution is stable for 1 month at -20 oC.

Protocol:
1. Suspend cells in Hanks·balanced salt solution (HBSS) to prepare 5-10 x 106 cells/ml medium.
2. Add Zinquin ethyl ester stock solution to the cell suspension to prepare 2.4 mM Zinquin ethyl ester (1/1000 vol of the cell suspension) as the final concentration.
3. Incubate the cell suspension at 37 oC for 30 min.
4. Wash the cells 3 times with HBSS, and then prepare 2-5 x 106 cells/ml of cell suspension.
5. Determine the fluorescence intensity of each cell using a fluorescence microscope or a confocal laser microscope coupled with an image analyzer.
* Cell staining conditions differ by cell type, so it is necessary to optimize the conditions for each experiment.