Created: July 24, 2017

For research use only

CaTM-3 AM
Table 1.

Product information

Catalog no.

Product nameAmountGC507

CaTM-3 AM

40 nmol × 5

Storage upon receipt

Stability≤−20°C, keep desiccated1 year (when unopenedand protected from light.

and stored as described.)

1. About CaTM-3
CaTM-3 AM is a fluorescent probe to detect intracellular calcium ion by red fluorescence at an emission
maximum of 609 nm corresponding to 590nm excitation. Its fluorescence intensity increases proportionally tothe calcium concentration. The reagent, acetoxymethyl ester (AM) of CaTM-3 is easily incorporated intocytoplasm and hydrolyzed by intracellular esterases. The product is suitable for quantitative analysis of calcium
ions because CaTM-3 usually distributes
equally in the cytoplasm.

Specifications
Absmax595 nmEmmax

609 nmKd (Ca2+)

0.19 μM

Φ0.37■ Storage

Fig. 1

The reagent is shipped as a dried powder in aSpectra of CaTM-3

nitrogen gas filled tube. Upon receipt, store at≤−20°C, keep desiccated and protected from light.
Dissolve the reagents to DMSO just before theuse. We provide no warranty for the reagents
which was stored as a solution.

2. Imaging protocols■ Materials required but not provided

Fig. 2.

・Dimethyl sulfoxide (DMSO)

concentrations

・Observation buffer (ex. Hank’s Balanced Salt
Solution, HBSS).

Fluorescence intensity against calcium ion

■ Preparation of reagent
1. CaTM-3 AM is pale yellowish brown solid. Before opening the vial, spin down the solid to the bottom by amicrocentrifuge or by a desktop centrifuge.
2. Warm the vial to the room temperature and add 40 μL of DMSO to one vial to make 1 mM stock solution,
which will be a pale blue solution. Dissolve the solid completely.

■ An example observation of intracellular calcium signals
1. Seed HeLa cells to a poly-ᴅ-lysine coated glass-bottomed dish in DMEM + 10% FBS (with 1% penicillinand 1% streptomycin), then culture at 37oC, 5% CO2 for >12 hours. Before loading the dye, remove theculture medium and rinse the cells three times with HBSS.
2. Dilute the stock solution of CaTM-3 AM with HBSS to make 3 μM staining solution. Add the solution to thedish to load the dye. Incubate at 37oC for 30 min.
3. Remove the staining solution and rinse the cells with HBSS three times.
4. Observe the cells in HBSS by fluorescence microscopy. See the ‘Fluorescence observation’ section forchoosing the excitation/emission conditions.
5. Stimulate the cells by adding histamine (final 1 μM) to the solution during the observation. You may
observe fluctuation of intracellular calcium concentration.
6. Add ionomysin (final 5 μM) to the solution. You may detect an increase in the intracellular calcium
concentration.
※ In case of cultured cells, fluorescence decrease has been observed by the addition of 0.01% Pluronic F127.

■ An example observation of calcium signals in a brain slice
1. Dissect a brain from a mouse at postnatal day 7 (P7) and prepare slices of hippocampus (thickness 300μm).
2. Mount the slice on a membrane filter in culture media and incubate in at 37oC, 5% CO2 for 7 days.
3. Replace the culture medium with 8 μM of CaTM-3 AM diluted in artificial cerebrospinal fluid (ACSF)
containing 0.01% Pluronic F-127, 0.005% Cremophor EL, and 0.08% DMSO as a cosolvent. Incubate at37oC for 40 minutes to load CaTM-3 AM to the slice.
4. Rinse the slice with ACSF for three times, and allowed to recover in 2 mL ACSF at 37oC for 45 minutes.
5. Transfer the slice into a recording chamber heated at 35oC and continuously perfused with ACSF.
Acquire images using confocal microscope, etc.
※ For brain slices, addition of 0.01% Pluronic F-127 and 0.005% Cremophor EL is preferable, whereas
addition of these reagents is not preferable for cultured cells.

■ Fluorescent observation
For confocal fluorescence microscopy, excite the dye with either 568 nm or 590 nm laser and observe thefluorescence ranging 610-680 nm. Alternatively, filter cube sets such as mCherry or TexasRed (for NIKON
microscopes), U-FYW or U-FMCHE (for OLYMPUS microscopes) are recommended for observation byfluorescence microscopy.

■ Reference

Hirabayashi K, Hanaoka K, Egawa T, Kobayashi C, Takahashi S, Komatsu T, Ueno T, Terai T, Ikegaya Y,Nagano T, Urano Y. (2016) Cell Calcium 60 256-265

Table 2. Related Products
Catalog no.

Product name

Major applications

GC401
GC402

CaSiR-1TM

GC403

CaSiR-1TM AM

GC404

CaSiR-1TM Assay Kit

GC501
GC502

CaTM-2™

Detection and live cell imaging of hypochlorous acid (HOCl).

GC503
GC504

CaTM-2™ AM

Detection of hydroxyl radical (・OH) and peroxynitrite (ONOO−).

GC505

CaTM-2™ Assay Kit

SK1003-01

NiSPY-3

SK1004-01

DAF-FM DA

SK1005-01

DAR-4M

SK1006-01

DAR-4M AM

Detection of ferrous ions (Fe2+) in Golgi.
Detection of Zn2+ ions.
For live cell imaging of Zn2+ ions.

Detection of hydroxyl radical ( ・ OH), peroxynitrite (ONOO−) andhypochlorous acid (HOCl).
Detection of peroxynitrite (ONOO−).
Detection of intracellular NO by green fluorescence.
Detection of NO by orange fluorescence.
Detection of intracellular NO by orange fluorescence.

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