Microsoft Word PK CA707 50016.doc

Transcript

1 Fluo-3 AM Solution (1 mM in DMSO) 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, acetomethyl ester Instruction Manual Catalog Number PK-CA707-5001 5 Description mum at 506 nm, thus ng its absorption maxi Fluo-3 is a long wavelength calcium probe havi ither the excitation nor making it excitable by the argon-ion laser. Un like Fura-2 and Indo-1, ne ⁺ the emission maximum of the sensor shifts significantly before and after Ca² binding. As a result, the ratioing technique is not applicable to Fluo-3. Fluo-3 is essentially nonfluorescent present, but the fluorescence incr eases at least 40 times on Ca² binding. Also, ⁺ ⁺ without Ca² -2 and Indo-1, it is more useful more weakly (higher Kd) than do Fura ⁺ because Fluo-3 binds Ca² ⁺ concentration during Ca² ⁺ spikes. The long wavelength of the for measuring high transient Ca² 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 es ter derivative of Fluo-3 that can be easily loaded into cells by , once inside the cells it is incubation. Fluo-3 AM itself doe s not respond to calcium. However readily hydrolyzed to Fluo-3 free acid by nonspecific esterases. Quantity 1.0 ml Excitation / Emission Maxima λ ⁺ ]); em (DNA) = 506/526 nm (low or high [Ca² ex\ λ ¹cm Extinction Coefficient: 86,000 M ¹ (506 nm) ⁻ ⁻ Molecular Structure O O COCH CH O 2 3 O Cl Cl CH 3 O CH OCH 2 2 OCCH (CH N ) COCH COC H OCCH ) N(CH 2 2 3 2 2 2 2 3 OO O O Molecular Weight / 1129.9 Da; C₅₁H₅₀Cl₂N₂O₂₃ Molecular Formula Purity >95% (as determined by HPLC) Appearance / Formulation / Orange red liquid. Solubility Intended Use diagnostic or therapeutic procedures. For in vitro research use only. Not for Storage & Stability Fluo-3 AM ester should be stored desiccated at –20°C upon receipt. We recommend DMSO as the solvent for making stock solution. To di ssolve the material, both DMSO and Fluo-3 AM xing. Allow sufficient time for the AM ester to should be warmed to room temperature before mi dissolve since the dissolution can be kinetically slow. For long-term storage, anhydrous DMSO should be used and the DMSO stock solution should be tightly sealed and frozen at –20°C. Also, the stock solution should be warmed to room temperature each time before opening to avoid moisture condensation, which may hydrolyze the AM ester during long term storage.

2 Applications The fluorescent calcium indicator Fluo-3 AM ester is a membrane-permeant form of Fluo-3 and can be loaded into most of cells by incubation wi th dilute aqueous solutions of the AM ester. The following procedure serves as an approximate guide for loading Fluo-3 AM into cells: ension or on a slide; a) prepare cells in susp b) prepare a 1-5 mM stock solution using anhydrous DMSO; c) dilute an aliquot of the DMSO stock solution to a concentration of 1-5 μM with a suitable buffer. The final concentration of the dye should be as low as possible in order to minimize PromoKine also offers Pluronic F-127 (PK- background fluorescence and nonspecific staining. CA707-59000/-59004/-59005) that facilitates AM ester solubilization. A 20% Pluronic F-127 for pure DMSO for making the AM ester stock solution in DMSO can be used to substitute solution if solubility problem occurs; d) mix equal volumes of aqueous AM ester and cell suspension and incubate for 15 to 60 minutes at 20°C to 37°C; e) wash the cells twice with buffer. For more detailed information on loading the dye, please refer to the literature. Calcium concentration and fluorescence ar e related according to the equation: [Ca² ] = Kd [(F (Fmin)/(Fmax ( F)] ⁺ where F is the fluorescence of the indicator at experimental calcium concentration, Fmin is the fluorescence in the absence of calcium and Fmax is the fluorescence of the indicator at saturated calcium concentration. The Kd for Fluo-3 was reported to be 450 nM in cell-free media (325 nM in defined buffer). However, the Kd is usually affected by a number of factors in cells including pH, proteins concentrations, ionic strength, temperature and viscos ity. Thus, calibration of the Kd is necessary for accurate measurement of intracellular calciu m concentrations. For more detailed information PromoKine offers A-23187 (PK-CA707-59001), an on calibration, please refer to the literature. ionophore that is commonly used for intra cellular calibration of calcium indicators. PromoKine also offers EDC (PK-CA707-59002, also known as EDAC), which can be used to fix calcium indicators in cells, if post histochemical studies are desired following physiological experiments. General Protocol (for Human T cells)* Reagents: - 2 mM Fluo 3-AM/DMSO (1 mg Fluo 3-AM in 442 μl DMSO) - Pluronic F127 - Hanks·balanced salt solution (HBSS) ₂ - HEPES buffer saline (10 mM HEPES, 1 mM Na HPO4, 137 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , , 5 mM glucose, 0.1% BSA, pH 7.4) ₂ 0.5 mM MgCl Protocol: AM/DMSO solution. Pluronic F127 prevents 1. Add 16.5 mg Pluronic F127 to Fluo 3- 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°C for 20 minutes. 4. Add 5 volumes of HBSS containing 1% fetal calf serum, and continue the incubation for another 40 minutes. 5. Wash the cells 3 times with HEPES buffer salin e. Then resuspend the cells to prepare 1x10 ⁵ cells/ml solution using HEPES buffer saline. 6. Incubate at 37°C for 10 minutes. 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. References 1) J. Biol. Chem. 264, 8171(1989) 2) J. Biol. Chem. 264, 8179(1989) 3) Meth. in Enzymol. 192, 38 (1990) Caution Potentially harmful. Avoid prolonged or repeated exposure. Avoid getting in eyes, on skin, or on clothing. Wash thoroughly after ha ndling. If eye or skin contact occurs, wash affected areas with plenty of water for 15 minutes and seek medical advice. In case of inhaling or swallowing, move individual to fresh air and seek medical advice immediately. PromoCell GmbH United Kingdom North America Phone: 0800 – 96 03 33 (toll free) 1 – 866 – 251 – 2860 (toll free) Phone: Sickingenstr. 63/65 Fax: 0800 – 169 85 54 (toll free) 1 – 866 – 827 – 9219 (toll free) Fax: 69126 Heidelberg Other Countries Deutschland Germany Phone: +49 6221 – 649 34 0 Telefon: 0800 – 776 66 23 (gebührenfrei) Fax: +49 6221 – 649 34 40 0800 – 100 83 06 (gebührenfrei) Fax: France Email: [email protected] 32 (ligne verte) Téléphone: 0800 90 93 www.promokine.info 0800 90 27 36 (ligne verte) Téléfax:

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