Fluo-2 MA Brighter, Faster, Cheaper

Transcription

1 Fluo-2 MA Brighter, Faster, Cheaper Fluo-2 MA Essentials A direct substitute for Fluo-3 and Fluo-4 The same molecule as Fluo-8 Nearly twice as bright as Fluo-4 Nearly four times as bright as Fluo-3 AM ester loads well at room temperature, faster than Fluo-3 and Fluo-4 Excitation at 490 nm, emission at 515 nm, and 390 nm K d Greater than two hundred-fold increase from zero to saturated calcium Why is it called Fluo-2? Potassium Salt product specifications AM ester product specifications Potassium Salt MSDS AM ester MSDS Performance Comparison of Fluo-2 MA, Fluo-3, Fluo-4, and Fluo-8 Images comparing Fluo indicators in rat embryo fibroblasts Bar graph quantitation of Fluo-indicator comparison Other Fluo-2 Variants Fluo-2 HA (High Affinity) Fluo-2 LA (Low Affinity) Fluo-2 LR (Leakage Resistant) Fluo-2 NM (Near Membrane) Fluo-2 Mito (Mitochondrial) Mag-Fluo-2 (Magnesium)

2 Why is it called Fluo-2? The three most popular long wavelength fluorescent calcium indicators; Fluo-2, Fluo-3, and Fluo-4; were invented by TEFLabs creator Akwasi Minta and by Nobel Laureate Roger Tsien at University of California Berkeley (US Patent , 1991). The inventors at first chose Fluo- 3 as the best indicator, but researchers found that Fluo-4 is twice as bright and loads more easily. Recent experiments have shown that Fluo-2 is the brightest of all, nearly twice as bright as Fluo- 4, and that its AM ester loads more readily into the cell. Other companies have renamed Fluo-2, for example as Fluo-8. In seeking transparency with our customers, we prefer to follow the original nomenclature: Fluo-2. FLUO FAMILY N(CH 2 CO 2 - ) 2 O O N(CH 2 CO 2 - ) 2 X X R O O O - X R Fluo-2 MA (Medium Affinity) H H Fluo-8 H H Fluo-2 HA (High Affinity) H Me Fluo-3 Cl Me Fluo-4 F Me

3 Product Specifications Product Description Product Name FLUO-2 MA (AM), US Patent # 5,049,673 (FLUO-2 Medium Affinity, Acetoxymethyl ester) Cat. # 0200, 0202 Structure Description Appearance MW Cell-permeable acetoxymethyl (AM) ester derivative of Fluo-2 MA. Approximately twice as bright as Fluo-4, four times Fluo-3 Red-orange glossy solid 1047g/mol K d 390 nm (After AM hydrolysis) Analysis TLC Solvent Ethyl Acetate R f 0.4 HPLC Column C 18 Detector Settings 254 nm, 450 nm Purity >95% 1H NMR Solvent All relevant peaks present Deuterated acetone Mass Spectrum Mass-to-Charge Ratio Electrospray Ionization 1047 M/Z

4 Absorbance Spectrum Solvent Ethyl Acetate λ max 452 ± 3 nm ε 26,000 M -1 cm -1 Emission Spectrum Solvent 10 mm EGTA, 100 mm KCl, 10 mm MOPS, ph 7.2 λ max 515 ± 3 nm (after AM hydrolysis) Excitation Spectrum Solvent 10 mm EGTA, 100 mm KCl, 10 mm MOPS, ph 7.2 λ max 490 ± 3 nm (after AM hydrolysis) Fluorescence and binding to calcium are observed only after hydrolysis by non-specific esterases to the Fluo-2 MA pentanion.

5 Product Specifications Product Description Product Name FLUO-2 MA (K + Salt), US Patent # 5,049,673 (FLUO-2 Medium Affinity, Pentapotassium Salt)) Catalog Number 0204 Structure Description Appearance MW K d Non-ratiometric calcium indicator exhibiting greater than two hundred-fold fluorescence enhancement from zero to saturated calcium Approximately twice as bright as Fluo-4, four times Fluo-3 Red-orange solid 877 g/mol 390 nm Analysis TLC C 18 Solvent 3:2 methanol / brine R f 0.5 HPLC Column C 18 Detector Settings 254 nm, 450 nm Purity >90% 1H NMR Solvent Mass Spectrum Mass-to-Charge Ratio All peaks present Deuterated water Electrospray Ionization 877 M/Z

6 Absorbance Spectrum Solvent 10 mm EGTA, 100 mm KCl, 10 mm MOPS, ph 7.2 λ max 490 ± 3 nm ε 26,000 M -1 cm -1 Emission Spectrum Solvent 10 mm EGTA, 100 mm KCl, 10 mm MOPS, ph 7.2 λ max 515 ± 3 nm Excitation Spectrum Solvent 10 mm EGTA, 100 mm KCl, 10 mm MOPS, ph 7.2 λ max 490 ± 3 nm

7 Catalog Number 0200 and 0202 Material Safety Data Sheet Product Name Physical Characteristics Fluo-2 MA (AM) Red solid gum Quantity 1 mg / unit (0200) or 20 x 50 μg / unit (0202) Odor None Fire and Explosion Hazards None Toxicology Data Emergency and First Aid Not known In case of contact with skin, eyes, or mouth, wash profusely with water. If swallowed, rinse mouth with water and seek medical advice. For Further Information TEF Labs, Inc. or in Emergency Contact 9415 Capitol View Dr. the Manufacturer Austin, TX (512) support@teflabs.com Safety Precautions and Control Measures Potentially harmful if inhaled or ingested. Do not get in eyes, on skin, or on clothing. Potential skin and eye irritant. Wash thoroughly after handling. Gloves should be worn when working with this material. Clean up procedure: wash with soap and water. This material is for research and experimental applications only. It is not intended for food, drug, household, agricultural, or cosmetic use. All materials should be handled only by technically qualified individuals experienced in handling potentially hazardous chemicals. The above is correct to the best of our knowledge. The user should make independent decisions regarding completeness of the information based on all sources available. TEF Labs, Inc. shall not be held liable for any damage resulting from handling or from contact with the above product. Date of writing or last revision: March 2010

8 Catalog Number 0204 Material Safety Data Sheet Product Name Physical Characteristics Quantity Odor Fluo-2 MA (K + Salt) Red solid 1 mg / unit None Fire and Explosion Hazards None Toxicology Data Emergency and First Aid Not known In case of contact with skin, eyes, or mouth, wash profusely with water. If swallowed, rinse mouth with water and seek medical advice. For Further Information TEF Labs, Inc. or in Emergency Contact 9415 Capitol View Dr. the Manufacturer Austin, TX (512) support@teflabs.com Safety Precautions and Control Measures Potentially harmful if inhaled or ingested. Do not get in eyes, on skin, or on clothing. Potential skin and eye irritant. Wash thoroughly after handling. Gloves should be worn when working with this material. Clean up procedure: wash with soap and water. This material is for research and experimental applications only. It is not intended for food, drug, household, agricultural, or cosmetic use. All materials should be handled only by technically qualified individuals experienced in handling potentially hazardous chemicals. The above is correct to the best of our knowledge. The user should make independent decisions regarding completeness of the information based on all sources available. TEF Labs, Inc. shall not be held liable for any damage resulting from handling or from contact with the above product. Date of writing or last revision: March 2010

9 Images Comparing Fluo Indicators in Rat Embryo Fibroblasts REF52 rat embryo fibroblasts, cultured on No. glass coverslips, were incubated for 45 minutes at room temperature with 5 µm of the AM ester of each indicator in serum-free DMEM containing 25 mm HEPES (ph 7.4). The coverslips were then rinsed in HBSS, mounted in round dishes and bathed with HBSS. Indicators were excited with 488-nm light from a monochromator; fluorescence images were acquired with a cooled CCD camera through a 40 objective with high numerical aperture (NA = 1.4). Cells were stimulated with 2 µm ionomycin to elicit a sharp rise in intracellular Ca 2+ concentration. The images acquired immediately before and after ionomycin addition are shown. The fluorescence change resulting from ionomycin application in each population of cells was also analyzed; the results are displayed in the bar graph. The result for each indicator is derived from analyzing cells.

10 Accreditation Fluo indicator comparison images and quantitative bar graphs were contributed by: JPY Kao Center for Biomedical Engineering & Technology University of Maryland, Baltimore Medical Biotechnology Center University of Maryland Biotechnology Institute