Fluo-8 AM Non Wash Cell Calcium Flow Detection Kit

Fluo-8 AM Non Wash Cell Calcium Flow Detection KitThe components and specifications are as follows. Suggest using Fluo-8 cell loading reagents at a dosage of 100 μ L per well for 96 well plates and 25 μ L per well for 384 well plates. 96T, 960T, and 9600T can respectively complete experiments on 1, 10, and 100 96 hole or 384 hole plates.

1. Inoculate cells into transparent black cell culture plates at the bottom of 96 or 384 wells according to experimental requirements, with a serum concentration of no more than 10% in the cell culture medium. According to experimental requirements, 37 ℃ and 5% CO2Incubate in an incubator for a certain period of time.
2. Take out Fluo-8 loading buffer and QF (10x) reagent, equilibrate to room temperature, and mix thoroughly.
3. Remove Fluo-8 AM, dissolve at room temperature, centrifuge to collect the contents at the bottom of the tube, and place on ice. If Fluo-8 AM is not used all at once, it should be packaged and stored at -20 ℃ in the dark to avoid repeated freezing and thawing.
4. Calculate the required amount of Fluo-8 cell loading reagent by adding an equal volume of Fluo-8 cell loading reagent to each well and the culture medium inside the well
5. Fluo-8 cell loading reagent preparation: Dilute 10x QF reagent by 10 times and add it to Fluo-8 loading buffer. For example, add 1mL of 10x QF reagent to 9mL of Fluo-8 loading buffer and mix thoroughly. Fluo-8 AM is 400X, and the Fluo-8 loading buffer (containing 1x QF) prepared above is added at 1X. This is the Fluo-8 cell loading reagent.
6. Some cells, such as CHO cells, require the addition of Probenecid (CAS: 57-66-9) to Fluo-8 cell loading reagents to inhibit the transport of Fluo-8 out of the cell. Propionine sulfonate shu is provided by the customer themselves, and it is recommended to add Fluo-8 cell loading reagent at a final concentration of 2.5mM. The optimal concentration may need to be optimized for different cells.
Preparation of Propionine Sulfonate:Prepare 500 mM stock solution with 1N NaOH, dilute to 250 mM with 1X HBSS buffer before use, and add to Fluo-8 cell loading reagent, with a final concentration of 2.5 mM. (HBSS buffer is provided by the customer, with a formula of 20 mM HEPES buffer pH 7.4+1X Hank's Balanced Salt Solution, pH 7.4).
7. Standard method testing: Add an equal volume of Fluo-8 cell loading reagent to the culture medium in each well of the cell culture plate, for example, add 100 μ L of Fluo-8 cell loading reagent to 100 μ L of culture medium per well of the 96 well plate.
（Optional detection methods:The culture medium containing 10% FBS may decrease the reading value and increase the background. The culture medium in the well can be removed, and Fluo-8 cell loading reagent (prepared in steps 5 and 6) and HBSS buffer can be mixed in a 1:1 ratio. Add this mixture equivalent to twice the volume of the original culture medium for detection. Or use low concentration FBS culture medium for cell culture, and test according to the standard method in step 7
8. Incubate at 37 ℃ for 1 hour. The specific incubation temperature (such as room temperature or 37 ℃) and time (0.5hr-2hr or longer) need to be optimized according to the specific experiment.
9. Perform intracellular calcium flow detection: Set the detection parameters according to the experimental needs and detection equipment, add the test compound, and measure the fluorescence intensity by reading the bottom. Applicable testing equipment includes FlexStation, FLIPR (Molecular Devices), etc. FlexStation 3 reads fluorescence using Ex/Im=485/525 nm.

1. It is not recommended to mix different batches.
2. Without verification, it is not recommended to change the dosage of the detection reagent.