Screen Quest™ Fura-2 No Wash Calcium Assay Kit

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Unit Size: Cat No: Price (USD): Qty:
36320 $395


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

PlatformsFluorescence microplate reader, FDSS, NOVOStar, FlexStation, FLIPR
Storage Freeze (<-15 °C)
Minimize light exposure
Category Calcium,
Membrane and Channels,
Biomolecule Detection
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). This ratiometric calcium assay kit allows homogeneous measurement of intracellular calcium changes caused by activation of G-protein-coupled receptors or calcium channels. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fura-2 AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fura-2 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells and its fluorescence wavelength is blue-shifted upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which greatly increase the fluorescence intensity ofFura-2 at the short wavelength. The ratiometric characteristics of Fura-2 make this kit an ideal tool for more accurate measurement of cellular calcium concentration compared to Fluo-4 of the single wavelength. With a single addition, the assay is easy to perform and desirable in a high thoroughput environment. The assay can be used in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells in growth medium
  2. Add Fura-2 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at room temperature for 1-2 hour
  4. Monitor fluorescence intensity at Ex/Em = 340/510 nm and 380/510 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:340/380 nm
Emission:510 nm
Cutoff:470 nm
Recommended plate:Solid black
Other Instruments: FDSS, NOVOStar, FlexStation, FLIPR
Preparation of stock solution
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.

1. Fura-2 AM stock solution:
Add 200 µL of DMSO into the vial of Fura-2 AM (Component A), and mix them well. Note: 20 µL of Fura-2 AM stock solution is enough for one plate. Un-used Fura-2 AM stock solution can be aliquoted and stored at < -20 oC for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

2. Assay Buffer (1X):
a) For Cat. # 36320 (10 plates kit), make 1X assay buffer by adding 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B), and mix them well.
b) For Cat. # 36321 (100 plates kit), make 1X assay buffer by adding 90 mL of HHBS (Not included) into 10X Pluronic® F127 Plus (10 mL, Component B), and mix them well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 1X assay buffer at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles

Preparation of working solution

Fura-2 AM dye-loading solution:
Add 20 µL of Fura-2 AM stock solution into 10 mL of 1X assay buffer, and mix them well. Note: This working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fura-2 AM dye-loading solution into the cell plate. Note: If your compounds interfere with the serum, then it is important to replace the growth medium with HHBS buffer.

  2. Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 20 minutes. Note: If the assay requires 37 oC, perform the experiment immediately without further room temperature incubation.

  3. Prepare the compound plate with HHBS or your desired buffer.

  4. Run the calcium flux assay by monitoring the fluorescence increase at Ex/Em = 340/510 nm and 380/510 nm. Note: It is important to run the signal test before the experiment. Different instruments have their own intensity range. Note: For assays performed on FDSS, use the standard filters for Fura-2 calcium assays on the instrument.
Example data analysis and figures

The reading (Response 1) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Concentration samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator

Figure 1. ATP dose response in CHO-K1 cells measured with Screen Quest™ Fura-2 No Wash Calcium Assay Kit. CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour at room temperature. ATP (50 µL/well) was added by a FlexStation (Molecular Devices) to achieve the final indicated concentrations.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

Calcium imaging of cortical neurons using Fura-2 AM
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)

Measurement of [Ca2+]i in whole cell suspensions using fura-2
Authors: Hirst RA, Harrison C, Hirota K, Lambert DG.
Journal: Methods Mol Biol (2006): 37

[Load of calcium probe Fura -2/AM in Escherichia coli cells]
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805

Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219

Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica
Authors: Froschauer EM, Kolisek M, Dieterich F, Schweigel M, Schweyen RJ.
Journal: FEMS Microbiol Lett (2004): 49

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2
Authors: Berkova Z, Morris AP, Estes MK.
Journal: Cell Calcium (2003): 55

AMPA-induced Ca(2+) influx in cultured rat cortical nonpyramidal neurones: pharmacological characterization using fura-2 microfluorimetry
Authors: Fischer W, Franke H, Scheibler P, Allgaier C, Illes P.
Journal: Eur J Pharmacol (2002): 53

Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy
Authors: Novak I, Nitschke R, Amstrup J.
Journal: Cell Physiol Biochem (2002): 83

Measurement of [Ca2+]i in whole cell suspensions using fura-2
Authors: Hirst RA, Harrison C, Hirota K, Lambert DG.
Journal: Methods Mol Biol (1999): 31


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