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Screen Quest™ Fura-2 No Wash Calcium Assay Kit
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 of Fura-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.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells in growth medium
- Add Fura-2 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at room temperature for 1-2 hour
- Monitor fluorescence intensity at Ex/Em = 340/510 nm and 380/510 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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.
Note 20 µL of Fura-2 AM stock solution is enough for one plate. Unused 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.
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.
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. Unused 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.
SAMPLE EXPERIMENTAL PROTOCOL
- 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.
- 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.
- Prepare the compound plate with HHBS or your desired buffer.
- 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.
Spectrum
Citations
View all 1 citations: Citation Explorer
Etablierung von zellbasierten Assays zur Identifizierung von Inhibitoren des Chemokins CXCL8
Authors: J{\"o}st, Marina
Journal: (2016)
Authors: J{\"o}st, Marina
Journal: (2016)
References
View all 119 references: Citation Explorer
Calcium imaging of cortical neurons using Fura-2 AM
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)
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
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
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
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
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
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
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