Calcium Flux Assay Kit (Fluo-8, No Wash) ab112129 is a no-wash, fluorescence-based assay for detecting intracellular calcium mobilization in cells from mammals and other species.
Fluo-8 Calcium Flux Assay Kit - No Wash designed for success:
- Ideal indicator for measurement of cellular calcium because of its long wavelength, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium)
- Easy to use and fast protocol with no wash step required
- Can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation
- Cited in > 50 publications
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Ca++, Ca2+
Calcium Flux Assay Kit (Fluo-8, No Wash) ab112129 is a no-wash, fluorescence-based assay for detecting intracellular calcium mobilization in cells from mammals and other species.
Fluo-8 Calcium Flux Assay Kit - No Wash designed for success:
- Ideal indicator for measurement of cellular calcium because of its long wavelength, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium)
- Easy to use and fast protocol with no wash step required
- Can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation
- Cited in > 50 publications
How the assay works
In the Fluo-8 Calcium Flux Assay Kit protocol, cells are pre-loaded with Fluo-8 which can cross the cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-8 are cleaved by an esterase, resulting in a negatively charged fluorescent dye that stays inside the cell. Its fluorescence is greatly enhanced upon binding to calcium.If intracellular calcium levels increase, the fluorescence of Fluo-8 is significantly increased.
The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement make Fluo-8 the brightest green calcium indicator available, and it is an ideal tool for the measurement of cellular calcium through HTS screening.
Fluo-8 Calcium Flux Assay Kit protocol Summary
1. Prepare cells in growth medium with 1-5% FBS
2. Add Fluo-8 NW 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 hour
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm
Fluo-8 products available
Fluo-8 Calcium Flux Assay Kits are available in No Wash and Medium Removal variants:
- Calcium Flux Assay Kit (Fluo-8, No Wash) ab112129 is generally preferred as the workflow is simpler. It is also recommended for cell lines that are only weakly adherent and risk losing adherence when washing.
- Medium removal kit Fluo-8 Calcium Flux Assay Kit Fluo-8 Calcium Assay Kit - Medium Removal ab112128 is recommended for the testing of compounds, receptors and calcium channels that are sensitive to the presence of cell culture media or serum. Alternatively, for cells that can tolerate it, consider using a low serum % in culture media and use Calcium Flux Assay Kit (Fluo-8, No Wash) ab112129.
Fluo-8 is also available as a standalone biochemical Fluo-8 AM, green fluorescent calcium binding dye ab142773.
This product is intended to be used for monitoring calcium fluctuations in vivo in live cells using the following HTS imaging plate readers: FLIPR™, FDSS, BMG NOVOstar™, FLexStation, ViewLux, IN Cell Analyzer or Arrayscan.
Readers with an in built pipetter are essential for measuring calcium response accurately due to transient property of the calcium signal. Depending on dose of the agonist, the peak response is few seconds to 20 seconds, so one will need to add the compound by instrument and read the fluorescent signal simultaneously every second for ~1-2 min.
If you would like to quantify calcium concentration in vitro using cell extracts, we recommend using Calcium Detection Kit (Colorimetric) (Calcium Assay Kit (Colorimetric) ab102505) or Calcium Quantification Assay Kit (Calcium Assay kit (Fluorometric) ab112115).
How other researchers are using
Fluo-8 Calcium Flux Assay Kit - No Wash has been used in a variety of sample type including:
- Human OA chondrocytes and C28I2 cells 1
-Neuronal cells 2
- Human U2OS, HeLa, BJ, BJ-5ta (BJ-T), T98G, U87 MG and MRC5 cell lines and mouse embryonic fibroblasts (MEF) 3
References:
1- Fu W et al. 2024; 2- Lui L et al. 2023; 3- Munk S et al. 2023
Related and recommended products
See other alternative kits to quantify calcium concentration in vitro using cell extracts:
- Calcium Detection Kit (Colorimetric) Calcium Assay Kit (Colorimetric) ab102505
- Calcium Quantification Assay Kit Calcium Assay kit (Fluorometric) ab112115.
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Terms & Conditions.
Carbachol Dose Response was measured in HEK293 cells with ab112129 and a Fluo-4 calcium assay kit. HEK293 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using the ab112129 or the Fluo-4 kit for 1 hour at room temperature. Carbachol (50 µL/well) was added to achieve the final concentrations indicated.
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