Screen Quest™ Fluo-4 No Wash Calcium Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Extinction coefficient (cm -1 M -1) | 82000 |
Excitation (nm) | 495 |
Emission (nm) | 528 |
Quantum yield | 0.161 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit |
Overview | SDSProtocol |
Extinction coefficient (cm -1 M -1) 82000 | Excitation (nm) 495 | Emission (nm) 528 | Quantum yield 0.161 |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525nm |
Cutoff | 515 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Other instruments
ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLuxComponents
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Add Fluo-4 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at 37°C for 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
Important notes
Do not add additional probenecid. Thaw all the kit components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. Fluo-4 AM stock solution:
Add 200 µL of DMSO into the vial of Fluo-4 AM (Component A), and mix them well. Note: 20 µL of Fluo-4 NW stock solution is enough for one plate. Unused Fluo-4 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):
Make 1X Assay Buffer by adding 1 mL of 10X Pluronic® F127 Plus (10 mL, Component B) into 9 mL of HHBS buffer (Component C) and mix well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 10X Pluronic® F127 Plus (Component B) at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
Fluo-4 AM dye-loading solution:
Add 20 µL of Fluo-4 NW stock solution into 10 mL of 1X Assay Buffer and mix well. 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
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-4 AM dye-loading solution into the cell plate.
- Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 15 to 30 minutes. Note: If the assay requires 37°C, 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 intensity at Ex/Em = 490/525 nm.
Spectrum
Spectral properties
Extinction coefficient (cm -1 M -1) | 82000 |
Excitation (nm) | 495 |
Emission (nm) | 528 |
Quantum yield | 0.161 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal* | 523 | 551 | 0.11 |
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit | 523 | 551 | 0.11 |
Images
Citations
Authors: Drzazga, Anna and Kristinsson, Hjalti and Salaga, Maciej and Zatorski, Hubert and Koziolkiewicz, Maria and Gendaszewska-Darmach, Edyta and Bergsten, Peter
Journal: Molecular and Cellular Endocrinology (2017)
Application notes
The Eight Best Green Fluorescent Calcium Indicators
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Evaluation of FLIPR Calcium Assays for Screening GPCR and Calcium Channel Targets
Fluorescent Dye AM Esters
FAQ
Why is my Fluo-4 signal not at a useful magnitude?
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
How do I make an AM ester stock solution?
How do you measure Ca2+ response with calcium indicators like Calbryte 520, potassium salt?