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 | The unopened buffer solution should be stored at 4℃. Before use, it needs to be taken out and allowed to equilibrate at room temperature for 30 minutes, then inverted and mixed several times. The aliquoted buffer solution or 1x working solution can be stored at room temperature (22℃) for 1 week. The Fluo-8 reagent should be stored at -20℃ or -80℃ and will remain stable for 1 year. |
Background
Many vital intracellular biological reactions involve calcium flux. Detecting transient changes in intracellular calcium flux is crucial for studying intracellular biological processes and developing targeted drugs. The calcium-specific fluorescent dye method is the most commonly used technique for detecting intracellular calcium flux.
As a new generation calcium flux detection dye, Fluo-8 improves cellular loading and calcium response while maintaining the spectral wavelengths of Fluo-3 and Fluo-4 (Ex/Em ≈ 490/520 nm). Fluo-8 AM can be loaded into cells at room temperature, whereas Fluo-3 AM and Fluo-4 AM require 37°C for cell loading. Additionally, Fluo-8 AM is twice as bright as Fluo-4 AM and four times brighter than Fluo-3 AM.
In cellular loading and calcium flux analysis, calcium flux dyes often require cell washing, which significantly increases workflow during detection. Since some cells may detach during washing, this also introduces variability in the analysis. Therefore, no-wash dyes and methods represent a current trend in development.
To reduce dye efflux from cells, the anion transport protein inhibitor probenecid needs to be added to both the dye loading solution and subsequent analysis solutions.
Components
The contents of the kit include Fluo-8 reagent and buffer solution, which are separately filled in 1 ml spiral tubes and 10 ml or 100 ml brown bottles. The specifications are as follows:
Size |
Fluo-8 regent |
Buffer |
The number of detectable wells in a 96-well plate |
The number of detectable wells in a 384-well plate |
100tests |
100μl |
10ml |
200 wells |
1,000 wells |
1000tests |
1ml |
100ml |
2,000 wells |
10,000 wells |
10000tests |
10x1ml |
10x100ml |
20,000 wells |
100,000 wells |
Protocol
1. Cell Preparation
a) Digest cells cultured in normal medium with trypsin, add complete medium, count the cells, take the required total number of cells, and centrifuge. The following are suggested quantities for a 96-well plate; adjust accordingly for a 384-well plate. For a 96-well culture plate, it is generally recommended to use approximately 20,000 cells per well.
b) Resuspend the centrifuged cells in Hank’s salt solution or a similar phenol red-free culture medium supplemented with 1% fetal bovine serum. Then seed the cells into the culture plate at 50μl per well. Culture the cells for 3-5 hours until adherence or overnight.
2. Dye Loading
a) Take out the buffer and equilibrate at room temperature for 30 minutes, inverting and mixing several times. If the buffer is labeled as a 10x concentrate, dilute it to a 1x working buffer using deionized water.
b) Take out the Fluo-8 reagent and equilibrate at room temperature for 5-10 minutes, then centrifuge to collect the contents at the bottom of the tube. If the reagent is not used all at once, it can be aliquoted and stored at -20°C to minimize freeze-thaw cycles. Keep protected from light.
c) Calculate the required volume of dye loading solution based on 50μl per well. Add the Fluo-8 reagent to the 1x working buffer and mix thoroughly. The recommended starting dilution for Fluo-8 reagent is 1:100 in the working buffer, which can be adjusted based on cell type and requirements.
d) Calculate the required volume of probenecid stock solution and add it to the Fluo-8 working buffer, mixing well to prepare the loading solution.
e) Aspirate the culture medium from the wells of the culture plate and add the loading solution at 50μl per well. Gently shake for 10 seconds, cover, and incubate at 37°C for 1 hour.
3. Intracellular Calcium Flux Detection
a) Prepare compounds and agonists: It is recommended to use at least 6 concentration gradients for agonists. Include control wells without agonists or compounds in each reaction plate. The volume for agonist/compound plates is 100-200μl per well.
b) Prepare specialized pipette tip boxes compatible with the detection instrument.
c) Prepare the detection buffer: The detection buffer is Hank’s salt buffer supplemented with probenecid.
d) Aspirate the dye loading solution from the wells of the culture plate and add 100μl of detection buffer per well.
e) Set up the instrument detection program and relevant parameters, with excitation/emission wavelengths at 490/525nm. Read every 1-3 seconds for 2 minutes continuously.
f) Take a baseline reading before adding agonists/compounds.
g) Set up the corresponding positions of the agonist/compound plate wells and tips relative to the experimental cells.
h) Initiate the detection program for adding agonists/compounds and collect experimental data.
i) Perform data analysis using the (F-F0)/F0 *100% method or other validated methods.
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Bioactivity
