Asante NaTRIUM Green 1 (ANG-1)

Transcription

1 Page 1 of 10 Asante NaTRIUM Green 1 (ANG-1) ANG-1 is to Sodium as Fluo-4 is to Calcium ANG-1 Highlights Compatible with green argon laser, LED excitation, or two-photon IR laser excitation Twenty-fold enhancement in emission at 540 nm from zero to saturated sodium Sensitive and calibratable response to cytosolic sodium changes AM ester loads readily at room temperature Little compartmentalization, even distribution throughout the cytosol Good retention within cell at 37 C Resistant to photobleaching at 37 C Excitation maximum at 517 nm, emission maximum at 540 nm 81 mm Kd for sodium (in the absence of potassium) Potassium Salt product specifications AM ester product specifications Potassium Salt MSDS AM ester MSDS Experimental Results for ANG-1 Excitation and emission spectra of a titration with sodium chloride Images of ANG-1 response to TRPV1 channel agonization Images of ANG-1 response to gramicidin-induced sodium increase Images of ANG-1 response to ouabain inhibition of sodium pump ANG-1 (AM) Loading Protocols Acetoxymethyl ester loading protocol for ANG-1

2 Page 2 of 10 Product Specifications Product Name Asante Natrium Green 1 (K + Salt), or ANG-1 (K + Salt) Catalog Number 3520 or 3550 Description A non-ratiometric green emission sodium indicator Structure Patent Pending Molecular Weight 1000 g/mol Kd for Na + 81 mm (in the absence of K + ) Handling and Storage Shelf Life Store at -20 C. Protect from light and moisture Valid for one year after delivery, if stored properly Specification TLC C18 silica Solvent 7:3 methanol / 3M sodium acetate Rf 0.5 HPLC Column C18 Detector Settings 254 nm, 475 nm Purity >85% 1 H NMR All relevant peaks present Solvent Deuterated water Absorbance Spectrum Solvent Methanol λ max 523 ± 3 nm ε 85,000 M -1 cm -1 Emission Spectrum Solvent 135 mm NaCl, 10 mm MOPS, ph 7.2 λ max 540 nm ± 3 nm Excitation Spectrum Solvent 135 mm NaCl, 10 mm MOPS, ph 7.2 λ max 517 ± 3 nm

3 Page 3 of 10 Product Specifications Product Name Asante Natrium Green 1 (AM), or ANG-1 (AM) Catalog Number 3500, 3510, 3530, or 3540 Description Membrane permeable acetoxymethyl (AM) ester derivative of Asante Natrium Green 1, a non-ratiometric green emission sodium indicator Structure Patent Pending Molecular Weight 1100 g/mol Kd for Na + 81 mm (in the absence of K + ) Handling and Storage Shelf Life Store at -20 C. Protect from light and moisture Valid for one year after delivery, if stored properly Specification TLC Solvent 4% methanol in chloroform Rf 0.5 HPLC Column C18 Detector Settings 254 nm, 460 nm Purity >85% 1 H NMR All relevant peaks present Solvent Deuterated acetone Absorbance Spectrum Solvent Methanol λ max 469 ± 3 nm ε 25,000 M -1 cm -1 Emission Spectrum Solvent λ max Excitation Spectrum Solvent λ max Methanol 545 nm ± 3 nm Methanol 525 ± 3 nm

4 Page 4 of 10 Product Name Catalog Number 3520 Unit Size Molecular Weight Odor Fire and Explosion Hazards Handling and Storage Toxicology Data Emergency and First Aid Material Safety Data Sheet Asante Natrium Green 1 (K + Salt) 250 µg/unit 1000 g/mol None None Store at -20 o C. Protect from light and moisture 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 or in Emergency Contact the Manufacturer Texas Fluorescence Labs, Inc 9415 Capitol View Drive Austin, TX (512) probes@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. Texas Fluorescence Labs, Inc. shall not be held liable for any damage resulting from handling or from contact with the above product.

5 Page 5 of 10 Product Name Catalog Number 3500 Unit Size Molecular Weight Odor Fire and Explosion Hazards Handling and Storage Toxicology Data Emergency and First Aid Material Safety Data Sheet Asante Natrium Green 1 (AM) 500 µg/unit 1200 g/mol None None Store at -20 o C. Protect from light and moisture 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 or in Emergency Contact the Manufacturer Texas Fluorescence Labs, Inc 9415 Capitol View Drive Austin, TX (512) probes@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. Texas Fluorescence Labs, Inc. shall not be held liable for any damage resulting from handling or from contact with the above product.

6 fluorescence intensity fluorescence intensity ANG-1 Page 6 of Asante Natrium Green 1 Excitation Traces 967 mm NaCl 135 mm NaCl 94.5 mm NaCl 54 mm NaCl 27 mm NaCl 13.5 mm NaCl 6.75 mm NaCl 2.7 mm NaCl 1.35 mm NaCl 0 mm NaCl Lot 009a Emission at 540 nm wavelength (nm) Asante Natrium Green 1 Fluorimetric Titration: Equal amounts of ANG-1 (K + Salt), Lot 009a were dissolved in Buffers A (135 mm TMACl, 10 mm MOPS, ph 7.2) and B (135 mm NaCl, 10 mm MOPS, ph 7.2). Appropriate mixtures of these buffers gave the desired sodium concentrations. Solid NaCl was added to Buffer B to give a saturating concentration of 967 mm; no corrections for the increased ionic strength of the solution were applied. Excitation spectra were acquired with emission at 540 nm. Emission spectra were acquired with excitation at 517 nm. Asante Natrium Green 1 Emission Traces Lot 009a Excitation at 517 nm 967 mm NaCl 135 mm NaCl 94.5 mm NaCl 54 mm NaCl 27 mm NaCl 13.5 mm NaCl 6.75 mm NaCl 2.7 mm NaCl 1.35 mm NaCl 0 mm NaCl wavelength (nm)

7 Page 7 of 10 ANG-1 (AM) Response to TRPV1 Channel Agonization by Capsaicin in HEK293 Cells Figure 2a: Plot quantifying the enhanced fluorescence of ANG-1, resulting from increased cytosolic sodium concentration provoked by capsaicin activation of TRPV1 channels. Figure 2b: ANG-1 fluorescence before (left image, point A in Figure 2a) and after (right image, point B in Figure 2a) capsaicin activation of TRPV1 channels. TRPV1 is the natural receptor for vanilloid molecules like capsaicin. Once activated, it permits an influx of cations, including sodium and calcium, into the cell. ANG-1 responds to the influx of sodium over resting sodium levels with a nearly 2.5-fold increase in fluorescence (F/F0), as quantified in Figure 2a and imaged in Figure 2b. Technical Details: HEK293 cells permanently overexpressing TRPV1 were loaded min at room temperature with 5 µm ANG-1 (AM) + 75 ppm Pluronic F-127 in HCO3- buffered DMEM containing 10% fetal bovine serum (FBS), under 5% CO2 atmosphere. Experiments were conducted in HBSS. Excitation occurred at 488 nm. Average response of 108 cells. Experimental results courtesy of Dr. JPY Kao, University of Maryland

8 Page 8 of 10 ANG-1 (AM) Response to Monovalent Cation Ionophore Gramicidin in REF52 Cells Figure 3a: Plot quantifying the increase in the fluorescence of ANG-1, resulting from increased cytosolic sodium concentration brought about by the monovalent cation ionophore gramicidin. Figure 3b: ANG-1 fluorescence before (left image, point A in Figure 3a) and after (right image, point B in Figure 3a) sodium influx due to gramicidin application. Gramicidin acts as a channel for inorganic monovalent cations. ANG-1 in REF52 cells responds to the gramicidin-induced influx of sodium over resting sodium levels with a nearly 3.5-fold increase in fluorescence, as quantified in Figure 3a and imaged in Figure 3b. Technical Details: REF52 cells were loaded for 1-2 hours at room temperature with 5 µm ANG-1 (AM) + 75 ppm Pluronic F-127 in HCO3-buffered DMEM containing 10% fetal bovine serum (FBS), under 5% CO2 atmosphere. Experiments were conducted in HBSS. Excitation occurred at 488 nm. Each trace is the average response of cells. Cells were used immediately after loading. Experimental results courtesy of Dr. JPY Kao, University of Maryland

9 Page 9 of 10 ANG-1 (AM) Response to Ouabain Inhibition of the Sodium Pump in Astrocytes Figure 1a: Gray scale image of astrocyte loaded with ANG-1 (AM) Figure 1b: False color image of astrocyte loaded with ANG-1 (AM), prior to sodium pump inhibition by ouabain. Figure 1c: False color image of astrocyte loaded with ANG-1 (AM), during sodium pump inhibition by ouabain. Ouabain inhibits the sodium pump of living cells, causing cytosolic sodium concentration to rise. ANG-1 (AM) responds to the increased sodium concentration with increased fluorescence, as demonstrated in the fluorescence change from Figure 1b to Figure 1c. Experimental results courtesy of Dr. Jean-Yves Chatton, coordinator of the Cellular Imaging Facility UNIL-CHUV.

10 Page 10 of 10 ANG-1 (AM) Loading Protocol The following is supplied as a starting point for non-invasive loading of ANG-1 via the acetoxymethyl (AM) ester version. Due to cell type and other experimental variations, loading conditions will require optimization. 1. Prepare a 1 mm stock solution of ANG-1 (AM) in anhydrous dimethylsulfoxide (DMSO). For a 500 µg vial, dissolve the contents of the vial in 450 µl DMSO. For a 50 µg vial, use 45 µl DMSO. 2. Divide the stock solution into aliquots that will be consumed per experiment. Store the aliquots at -20 ºC, protected from moisture and light. Repeated freezing and thawing of a stock solution typically leads to degradation of the product via hydrolysis of the AM esters For your convenience, we supply ANG-1 (AM) in 50 µg packages. 3. Dilute the stock solution to twice the original volume with a solution of 20% Pluronic F-127 in DMSO. 4. Disperse the ANG-1/Pluronic F-127 solution into 100 times the volume of serumfree culture medium. The final ANG-1 (AM) concentration is 5 µm. (The concentration of ANG-1 AM an important variable requiring optimization.) Pluronic F-127 is 0.1% of the final solution. 5. Incubate the cells for one to two hours at room temperature. (Incubation time and temperature also require optimization.) 6. Remove the cell loading medium and wash the cells with serum-free and dyefree medium. 7. The cells are now loaded with ANG-1 (AM) and ready for your sodium imaging experiments.