Screen Quest™ Fluo-8 No Wash Calcium Assay Kit
Price | |
Catalog Number | |
Unit Size | |
Quantity |
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 |
Correction Factor (260 nm) | 1.076 |
Correction Factor (280 nm) | 0.769 |
Extinction coefficient (cm -1 M -1) | 23430 |
Excitation (nm) | 495 |
Emission (nm) | 516 |
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 |
Overview | ![]() ![]() |
Correction Factor (260 nm) 1.076 | Correction Factor (280 nm) 0.769 | Extinction coefficient (cm -1 M -1) 23430 | Excitation (nm) 495 | Emission (nm) 516 | Quantum yield 0.161 |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 510 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 with 1-5% FBS
- Add Fluo-8 NW dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at room temperature for 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Fluo-8 NW stock solution:
Add 20 µL (for Cat. # 36314) or 200 µL (for Cat. # 36315 and # 36316) of DMSO into the vial of Fluo-8 NW (Component A), and mix them well. Note: 20 µL of Fluo-8 NW stock solution is enough for one plate. Un-used Fluo-8 NW 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. # 36314 (1 plate kit) and # 36315 (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. # 36316 (100 plates kit), make 1X assay buffer by adding the whole bottle of 10 X Pluronic® F127 Plus, (10 mL, Component B) into 90 mL of HHBS buffer (not included in the kit), 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
Fluo-8 NW dye-loading solution:
Add 20 µL of Fluo-8 NW 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
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8 NW dye-loading solution into the cell plate. [We offer 2 separate medium removal calcium assay kits (Cat.# 36308 and 36309) for those who prefer to keep the medium removal step].
- Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes. Note: If the assay requires 37 oC, perform the experiment immediately without further room temperature incubation. Note: If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1-2 hours (It is recommended that the incubation time be no longer than 2 hours).
- 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. Note: It is important to run the signal test before the experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum instrument intensity counts. For example, the maximum fluorescence intensity count for FLIPR-384 is 65,000, so the instrument settings should be adjusted to have the signal test intensity around 7,000 to 10,000.
Spectrum
![spectrum](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fspectra%2Ffluo_8.png&w=2048&q=50)
Spectral properties
Correction Factor (260 nm) | 1.076 |
Correction Factor (280 nm) | 0.769 |
Extinction coefficient (cm -1 M -1) | 23430 |
Excitation (nm) | 495 |
Emission (nm) | 516 |
Quantum yield | 0.161 |
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield |
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit | 495 | 528 | 82000 | 0.161 |
Images
![Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 No Wash Calcium Assay Kit and Fluo-4 NW Calcium Assay Kit. HEK-293 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 dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-no-wash-calcium-assay-kit%2Fgraph-for-screen-quest-fluo-8-no-wash-calcium-assay-kit_ASCBt.webp&w=3840&q=75)
![<strong>Generation of large cardiac tissue sheets (L-CTSs).</strong><br />(A) Optimal view of the L-CTS placed in a 10cm-sized UpCell dish. Scale bar = 1 mm. (B) (C) Calcium transient of L-CTSs. (B) Representative Fluo-8 image of L-CTS (see also S1 video) and region of interests (ROIs). Magnification ×100. (C) Chronologic intensity change of Fluo-8. Note that the peak timings of 4 different ROIs are almost same chronologically. Source: <strong>Human iPS cell-derived cardiac tissue sheets for functional restoration of infarcted porcine hearts</strong> by Masanosuke Ishigami et al., <em>PLOS</em>, Aug 2018.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-no-wash-calcium-assay-kit%2Ffigure-for-screen-quest-fluo-8-no-wash-calcium-assay-kit_rdWWo.jpg&w=3840&q=75)
(A) Optimal view of the L-CTS placed in a 10cm-sized UpCell dish. Scale bar = 1 mm. (B) (C) Calcium transient of L-CTSs. (B) Representative Fluo-8 image of L-CTS (see also S1 video) and region of interests (ROIs). Magnification ×100. (C) Chronologic intensity change of Fluo-8. Note that the peak timings of 4 different ROIs are almost same chronologically. Source: Human iPS cell-derived cardiac tissue sheets for functional restoration of infarcted porcine hearts by Masanosuke Ishigami et al., PLOS, Aug 2018.
![SLY-induced pore formation-dependent Ca<sup>2+</sup> influx triggers PNC formation. (A–C) rSLY induces Ca<sup>2+</sup> influx in human platelets. The purified platelets marked with Fluo-8 were resuspended in HBSS (with 2 mM Ca<sup>2+</sup>) and rSLY/rSLY<sup>P353V</sup> (1 μg/mL) or rSLY that was pretreated by cholesterol (10 μg/mL). The Ca<sup>2+</sup> influx in platelets was observed using an FV1000 confocal laser scanning microscope. The Ca<sup>2+</sup> influx in platelets was observed using an FV1000 confocal laser scanning microscope. The following mean fluorescence intensity (MFI) of Ca<sup>2+</sup> mobilization was recorded by Series Analysis (XY-T) software in FV1000. C1-C10, cell 1-cell 10. (D) The EGTA effect on rSLY-induced CD62P release from platelets in human blood was assessed by flow cytometry. (E) The EGTA (3 mM) effect on <em>S. suis</em> supernatant-induced PNC formation was detected by flow cytometry. THB and PBS are the negative controls for culture supernatant and proteins, respectively. EGTA was dissolved in H<sub>2</sub>O. Data in B and C are given as the mean ± SD of three independent experiments from three different blood donors. **<em>P</em> < 0.01; ns, no significance; Cho, cholesterol; rSLY, recombinant SLY; 05ZYH33, wild type strain; ∆sly, isogenic sly mutants; Sup, supernatant. Source: <strong>Suilysin-induced Platelet-Neutrophil Complexes Formation is Triggered by Pore Formation-dependent Calcium Influx </strong>by Zhang et al., <em>Scientific Reports</em>, Nov. 2016.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-no-wash-calcium-assay-kit%2Ffigure-for-screen-quest-fluo-8-no-wash-calcium-assay-kit_iDBte.jpg&w=3840&q=75)
![5-HT will attenuate Ca<sup>2+</sup> uptake under the normoxia condition. The fresh-made mitochondria were pre-incubated with Calcium indicator Quest Fluo-8<sup>TM</sup>, AM or membrane potential dye Rhodamine 123 for 15 min and washed three times using potassium chloride (KCl) media containing 5 mM succinate. After recording the baseline, the mitochondria were perfused with 10 uM Ca<sup>2+</sup> or 5 uM FCCP. (A) Serotonin hydrochloride (5-HT) decreases mitochondrial Ca<sup>2+</sup> uptake. (B) Serotonin creatinine sulfate (5-HT(H<sub>2</sub>SO<sub>4</sub>)) attenuate Ca<sup>2+</sup> uptake. (C) The percentage of Ca<sup>2+</sup> uptaken by mitochondria of 5-HT groups (n = 6) and 5-HT (H<sub>2</sub>SO<sub>4</sub>) groups (n = 6). (D) Ca<sup>2+</sup> induced mitochondrial Ca<sup>2+</sup> uptake can be inhibited by RUR (n = 5). (E) Transmitochondrial potential (measured with 5 nM Rh-123) in control and 100 uM 5-HT pre-incubated mitochondria. 5 uM FCCP (Protonophore trifluoromethoxy carbonyl cyanide phenylhydrazond) was added to depolarize the mitochondria (n = 3). Source: <strong>5-HTR3 and 5-HTR4 located on the mitochondrial membrane and functionally regulated mitochondrial functions </strong>by Wang et al., <em>Scientific Reports</em>, Nov. 2016.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-no-wash-calcium-assay-kit%2Ffigure-for-screen-quest-fluo-8-no-wash-calcium-assay-kit_KUaoS.jpg&w=3840&q=75)
Citations
Authors: Zhang, Nan and Sui, Yixuan and Jendrichovsky, Peter and Feng, Hemin and Shi, Heng and Zhang, Xu and Xu, Shenghui and Sun, Wenjian and Zhang, Huatang and Chen, Xi and others,
Journal: Alzheimer's Research \& Therapy (2024): 1--16
Authors: Hall, Daniel B and Kostyla, Caroline H and Hales, Laura M and Soliman, Tarik M
Journal: JBMR Plus (2024): ziae045
Authors: Hsu, Julia Chu-Ning and Tseng, Hsu-Wen and Chen, Chia-Hui and Lee, Tzong-Shyuan
Journal: Experimental Animals (2024): 23--0148
Authors: Pinheiro, In{\^e}s and Calo, Nicolas and Paolini-Bertrand, Marianne and Hartley, Oliver
Journal: (2023)
Authors: Zhang, Nan and Sui, Yixuan and Jendrichovsky, Peter and Feng, Hemin and Shi, Heng and Zhang, Xu and Xu, Shenghui and Sun, Wenjian and Zhang, Huatang and Chen, Xi and others,
Journal: (2023)
Authors: Korkus, Eliza and Szustak, Marcin and Madaj, Rafal and Chworos, Arkadiusz and Drzazga, Anna and Kozio{\l}kiewicz, Maria and D{\k{a}}browski, Grzegorz and Czaplicki, Sylwester and Konopka, Iwona and Gendaszewska-Darmach, Edyta
Journal: Food \& Function (2023)
Authors: Korkus, Eliza and Szustak, Marcin and D{\k{a}}browski, Grzegorz and Czaplicki, Sylwester and Kad{\l}ubowski, S{\l}awomir and Kozio{\l}kiewicz, Maria and Konopka, Iwona and Gendaszewska-Darmach, Edyta
Journal: NFS Journal (2023)
Authors: Froemke, Robert and Ahmed, Ismail and Liu, Jingjing and Gieniec, Krystyna and Bair-Marshall, Chloe and Adewakun, Ayomiposi and Hetzler, Belinda and Arp, Christopher and Khatri, Latika and Vanwalleghem, Gilles and others,
Journal: (2023)
Authors: Tian, Qingqing and Xing, Kunming and Liu, Yongshu and Wang, Qian and Sun, Haonan and Sun, Ying-Nan and Zhang, Shusheng
Journal: STAR Protocols (2023): 102115
Authors: Martino, Pieter and Sunkara, Raghava and Heitman, Nicholas and Rangl, Martina and Brown, Alexia and Saxena, Nivedita and Grisanti, Laura and Kohan, Donald and Yanagisawa, Masashi and Rendl, Michael
Journal: Nature Cell Biology (2023): 1--13
Application notes
Evaluation of FLIPR Calcium Assays for Screening GPCR and Calcium Channel Targets
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
FAQ
Are there any substitutes for probenecid in calcium assays?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?