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AAT Bioquest

Quin-2, AM *CAS 83104-85-2*

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Physical properties
Dissociation constant (Kd, nM)60
Molecular weight829.76
SolventDMSO
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


CAS
83104-85-2
Molecular weight
829.76
Dissociation constant (Kd, nM)
60
Quin-2 binds calcium tightly and resembles calcium chelator EGTA in ability to bind calcium much more tightly than magnesium. Binding of calcium causes large changes in ultraviolet absorption and fluorescence. The wavelengths of light that cause fluorescence when calcium is bound are longer than the wavelengths that cause fluorescence when it is not bound. When excited at two different wavelengths, the ratio of the fluorescence intensities at the two wavelengths gives the ratio of the concentrations of bound to free calcium. Free Quin-2 concentration can be measured precisely, so free calcium concentration can be calculated precisely. Quin-2 may be injected into cells to measure moment-to-moment changes in intracellular calcium concentration. Quin-2 AM is permeable to cells, and used for studying live cells.

Platform


Fluorescence microplate reader

Excitation340
Emission495
Cutoff475
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Quin-2 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Quin-2 AM in high-quality, anhydrous DMSO.   

PREPARATION OF WORKING SOLUTION

Quin-2 AM Working Solution
On the day of the experiment, either dissolve Quin-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Quin-2 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note     The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Quin-2 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note     If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solution, can be purchased from AAT Bioquest.

SAMPLE EXPERIMENTAL PROTOCOL

Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Quin-2 AM working solution into your cell plate.
    Note     If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
    Note     Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
  4. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
  5. Add the stimulant as desired and simultaneously measure fluorescence using a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em = 340/495 cutoff 475 nm. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Quin-2, AM *CAS 83104-85-2* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM120.517 µL602.584 µL1.205 mL6.026 mL12.052 mL
5 mM24.103 µL120.517 µL241.034 µL1.205 mL2.41 mL
10 mM12.052 µL60.258 µL120.517 µL602.584 µL1.205 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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References


View all 60 references: Citation Explorer
W-5 and quin 2-AM reverse the inhibitory effect of insulin on lipolysis due to dibutyryl cAMP
Authors: Goko H, Matsuoka A.
Journal: Diabetes Res Clin Pract (1999): 101
Calcium chelator Quin-2 prevents crocidolite-induced DNA strand breakage in human white blood cells
Authors: Faux SP, Michelangeli F, Levy LS.
Journal: Mutat Res (1994): 209
Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2
Authors: Lakowicz JR, Szmacinski H, Nowaczyk K, Lederer WJ, Kirby MS, Johnson ML.
Journal: Cell Calcium (1994): 7
Possible mechanisms of epinephrine actions in quin-2-loaded platelets refractory to arachidonic acid
Authors: Rao GH, Gerrard JM, Murthy M, White JG.
Journal: Biochem Med Metab Biol (1993): 322
Fluorescence lifetime imaging of calcium using Quin-2
Authors: Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML.
Journal: Cell Calcium (1992): 131
Toxicity to isolated hepatocytes caused by the intracellular calcium indicator, Quin 2
Authors: Carpenter-Deyo L, Duimstra JR, Hedstrom O, Reed DJ.
Journal: J Pharmacol Exp Ther (1991): 739
Involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes
Authors: Carpenter-Deyo L, Reed DJ.
Journal: J Pharmacol Exp Ther (1991): 747
Aspirin, prostaglandin E1 and Quin-2 AM-induced platelet dysfunction: restoration of function by noradrenalin
Authors: Rao GH, White JG.
Journal: Prostaglandins Leukot Essent Fatty Acids (1990): 141
Effect of Quin-2 on Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i
Authors: Blanco P, Martinez-Serrano A, Bogonez E, Satrustegui J.
Journal: Cell Calcium (1990): 25
Quin-2 and fura-2 measure calcium differently
Authors: Mazorow DL, Millar DB.
Journal: Anal Biochem (1990): 28