Hela cells were seeded in a 96-well cell culture plate at a density of 20,000 cells per well in 50 µL. Initially, the cells were suspended in Hank's Balanced Salt Solution (HBSS) supplemented with 1% Fetal Bovine Serum (FBS).
After the cells adhered to the plate, the medium was replaced with 50 µL per well of the Fluo-8 AM loading solution. The cells were then incubated for 1 hour at 37°C.
Following the incubation, the dye loading solution was aspirated. Each well was washed and replaced with 100 µL of assay buffer.
Calcium flux was induced and measured using a FlexStation 3 microplate reader. An agonist was added during the kinetic read, and fluorescence was monitored. Fluorescence readings (Excitation/Emission = 485/525 nm) were taken both before (baseline) and after agonist stimulation.
The data were analyzed, and the half-maximal effective concentration (EC50) was calculated.
Fluo-8免洗细胞钙流检测试剂盒
Fluo-8免洗细胞钙流检测试剂盒
Product Details
Product Details
Product Specification
| Synonyms | Fluo-8 No Wash Calcium Flux Assay Kit |
| Stability & Storage | Dry ice transport. After receiving the kit, store it protected from light at −20°C or lower. Following initial use, Fluo-8 loading buffer and QF (10×) reagent can be stored protected from light at 4°C for up to 1 month. Fluo-8 AM should be aliquoted and stored protected from light at −20°C or lower; for long-term storage (>1 month), storage at −80°C is recommended. |
Background
Detecting transient changes in intracellular calcium flux is crucial for studying cellular biological responses and developing targeted drugs. The calcium‑specific fluorescent dye method is the most commonly used approach for measuring intracellular calcium flux. Fluo‑8 AM, as a next‑generation calcium flux detection dye, enhances both cellular loading efficiency and calcium responsiveness, making it currently the most potent calcium‑specific fluorescent dye; its fluorescence is twice as bright as that of Fluo‑4 AM and four times brighter than Fluo‑3 AM. Youbo Biotech's Fluo‑8 AM wash‑free cell calcium flux assay kit eliminates the need for cell washing and removal of the Fluo‑8 loading reagent during cell loading and calcium flux detection, simplifying the experimental procedure and making it particularly well suited for high‑throughput compound screening.
Components
The components and specifications of the Fluo-8 AM wash-free cell calcium flux assay kit are as follows. It is recommended to use 100 μL per well for a 96-well plate and 25 μL per well for a 384-well plate when using the Fluo-8 cell-loading reagent. Kits containing 100, 1,000, and 10,000 tests can respectively perform experiments on 1, 10, and 100 plates of either 96-well or 384-well format.
Components |
100 tests |
1,000 tests |
10,000 tests |
Fluo-8 Loading Buffer |
10 mL |
100 mL |
10 × 100 mL |
Fluo-8 AM (400x) |
25 μL |
100 mL |
10 × 100 mL |
QF Reagent (10x) |
1 mL |
100 mL |
10 × 100 mL |
Protocol
1.According to experimental requirements, seed cells in 96-well or 384-well black-bottom transparent cell culture plates, with serum concentration in the culture medium not exceeding 10%. Depending on experimental needs, incubate at 37℃ 5% CO2in an incubator for a specified time.
2.Remove the Fluo-8 loading buffer and QF (10x) reagent, equilibrate to room temperature, and mix thoroughly.
3.Take out the Fluo-8 AM, allow it to thaw at room temperature, centrifuge to collect the contents at the bottom of the tube, and place it on ice. If the Fluo-8 AM is not used up at once, aliquot it, store it at -20℃ in the dark, and avoid repeated freeze-thaw cycles.
4.Calculate the required amount of Fluo-8 cell-loading reagent by adding to each well a volume equal to that of the culture medium already present in the well.
5.Fluo-8 cell loading reagent preparation: Dilute the 10x QF reagent by a factor of 10 into the Fluo-8 loading buffer; for example, add 1 mL of 10x QF reagent to 9 mL of Fluo-8 loading buffer and mix thoroughly. Fluo-8 AM is 400X, add it at a 1X concentration to the above-prepared Fluo-8 loading buffer (containing 1x QF); this is the Fluo-8 cell loading reagent.
6.Some cells, such as CHO cells, require probenecid (Probenecid, Cas57-66-9) to be added to the Fluo-8 cell loading reagent to inhibit the efflux of intracellularly loaded Fluo-8 from the cells.Probenecid is provided by the customer themselves.It is recommended to add probenecid to the Fluo-8 cell-loading reagent at a final concentration of 2.5 mM. For different cells, the optimal concentration may need to be adjusted.
Probenecid preparation: Prepare a 500 mM stock solution using 1N NaOH. Before use, dilute it with 1X HBSS buffer to 250 mM, then add it to the Fluo-8 cell-loading reagent to achieve a final concentration of 2.5 mM. (The customer needs to provide the HBSS buffer themselves,with the formula being 20 mM HEPES buffer pH 7.4 + 1X Hank's Balanced Salt Solution, pH 7.4).
7.Standard detection method:Add an equal volume of Fluo-8 cell-loading reagent to the culture medium in each well of the cell culture plate; for example, in a 96-well plate, add 100 μL of Fluo-8 cell-loading reagent to 100 μL of culture medium per well.
(Optional testing methods: Culture medium containing 10% FBS may reduce readings and increase background; it can be removed.In the wellsRemove the culture medium, mix Fluo-8 cell-loading reagent (prepared in steps 5 and 6) with HBSS buffer at a 1:1 ratio, and add this mixture to the wells—use a volume equivalent to twice the original medium—for detection. Alternatively, use culture medium with low concentrations of FBS for cell cultivation, and perform detection according to the standard method in step 7.)
8.37℃ Incubate for 1 hour. The specific incubation temperature (e.g., room temperature or 37℃) and time (0.5–2 hours or longer) should be optimized based on the particular experiment.
9.Perform intracellular calcium flux detection: Set detection parameters according to experimental needs and equipment, then add the test compound,Bottom readingDetect fluorescence intensity. Applicable detection equipment includesFlexStation, FLIPR (Molecular Devices), etc. Among them isFlexStation3 UseEx/Em = 485/525 nmRead fluorescence.
Guidelines
1. This product is intended for research use only.
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Bioactivity
