Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*
![Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW 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 growth medium was removed, and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines%2Fgraph-for-screen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines_rRo3Z.webp&w=640&q=75)
![Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW 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 growth medium was removed, and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines%2Fgraph-for-screen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines_rRo3Z.webp&w=640&q=75)
![Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay Kit and Fluo-4 NW 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 growth medium was removed, and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Carbachol using Fluo8 NW is about 1.2 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines%2Fgraph-for-screen-quest-fluo-8-medium-removal-calcium-assay-kit-optimized-for-difficult-cell-lines_rRo3Z.webp&w=128&q=25)
AT A GLANCE
Protocol summary
- Prepare cells
- Remove the growth medium
- Add Fluo-8 NW dye working solution
- Incubate at RT for 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
Important notes
Do not add additional probenecid. It is recommended to incubate the dye working solution no longer than 2 hours.
Thaw all components to room temperature before beginning protocol.
PREPARATION OF STOCK SOLUTION
1. Fluo-8 NW stock solution:
For Cat No. 36307, add 10 µL of DMSO into Fluo-8 NW (Component A), and mix them well.
For Cat No. 36308 and 36309, add 100 µL of DMSO into Fluo-8 NW (Component A), and mix them well. Note: 10 µL of Fluo-8 NW stock solution is enough for 1 plate.
2. Assay Buffer stock solution (1X):
For Cat No. 36307 and 36308, add 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B) and mix well.
For Cat No. 36309, add the whole bottle of 10X Pluronic® F127 Plus (10 mL, Component B) into 90 mL of HHBS buffer (not included in kit) and mix well. Note: 10 mL of 1X Assay Buffer is enough for one plate.
PREPARATION OF WORKING SOLUTION
Add 10 µL of Fluo-8 NW DMSO 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
- Remove the growth medium from the cell plate. Note: It is important to remove the growth medium in order to minimize background fluorescence and compound interference with serum or culture media. Note: Alternatively, grow the cells in growth medium with 0.5% - to 1% FBS to avoid medium removal step. In this case, 2X dye loading solution in HHBS buffer is needed. [We offer 2 separate no wash calcium assay kits (Cat No. 36315 and Cat No. 36316) for those who use 0.5% to 1% FBS in growth medium to avoid the medium removal step].
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Fluo-8 NW dye working solution into the cell plate.
- 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°C, 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 plates 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 your experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum intensity counts. For example, the maximum fluorescence intensity count for FLIPR-384 is 65,000, so the instrument setting should be adjusted to have its signal test intensity around 7,000 to 10,000.
Authors: Hsu, Julia Chu-Ning and Tseng, Hsu-Wen and Chen, Chia-Hui and Lee, Tzong-Shyuan
Journal: Experimental Animals (2024): 23--0148
Authors: Sun, Dawei and Sun, Yonglian and Janezic, Eric and Zhou, Tricia and Johnson, Matthew and Azumaya, Caleigh and Noreng, Sigrid and Chiu, Cecilia and Seki, Akiko and Arenzana, Teresita L and others,
Journal: Nature Communications (2023): 7940
Authors: Liu, An-Ran and Lin, Zhen-Jia and Wei, Ming and Tang, Yuan and Zhang, Hui and Peng, Xiang-Ge and Li, Ying and Zheng, Yu-Fan and Tan, Zhi and Zhou, Li-Jun and others,
Journal: The Journal of Headache and Pain (2023): 1--27
Authors: Wu, Chung-Kuan and Wu, Chia-Lin and Lee, Tzong-Shyuan and Kou, Yu Ru and Tarng, Der-Cherng
Journal: International journal of molecular sciences (2021): 2309
Authors: Quevedo, Ana C and Lynch, Iseult and Valsami-Jones, Eugenia
Journal: Environmental Science: Nano (2021)
Authors: Martin VV, Beierlein M, Morgan JL, Rothe A, Gee KR.
Journal: Cell Calcium (2004): 509
Authors: Bednar B, Cunningham ME, Kiss L, Cheng G, McCauley JA, Liverton NJ, Koblan KS.
Journal: J Neurosci Methods (2004): 247
Authors: do Ceu Monteiro M, Sansonetty F, Goncalves MJ, O'Connor JE.
Journal: Cytometry (1999): 302
Authors: Cheng H, Song LS, Shirokova N, Gonzalez A, Lakatta EG, Rios E, Stern MD.
Journal: Biophys J (1999): 606
Authors: Smith GD, Keizer JE, Stern MD, Lederer WJ, Cheng H.
Journal: Biophys J (1998): 15