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CAS:176767-94-5
Storage:Powder:-20℃，2 years;Insolvent(Mother Liquid):-20℃，6 months;-80℃，1 year
Purity:≥95%
Appearance:Solid
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This cell-permeant coumarin Ca2+ indicator BTC, AM exhibits a shift in excitation maximum from about 480 nm to 400 nm upon binding Ca2+, enabling ratiometric calcium measurements. Due to its high selectivity and low affinity for Ca2+ BTC is often used for the quantitation of high intracellular Ca2+levels. In addition, BTC, AM has also been used for monitoring potassium channel since thallium ion enhances the fluorescence of BTC.
Fluorescence microscope（for reference only）
Excitation FITC filter set
Emission FITC filter set
Recommended plate Black wall/clear bottom
Fluorescence microplate reader（for reference only）
Excitation 400, 480
Emission 540
Cutoff 515
Recommended plate Black wall/clear bottom
Instrument specification(s) Bottom read mode
Example protocol（For reference only）
BTC AM Stock Solution
Prepare a 2 to 5 mM stock solution of BTC AM in high-quality, anhydrous DMSO.
If it is necessary to prepare a stock solution, it is recommended to store in aliquots to avoid product failure caused by repeated freezing and thawing.
BTC AM Working Solution
On the day of the experiment, either dissolve BTC AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 μM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic F-127. For most cell lines, BTC AM at a final concentration of 4 to 5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note The nonionic detergent Pluronic F-127 is sometimes used to increase the aqueous solubility of BTC AM.
Note If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators.
SAMPLE EXPERIMENTAL PROTOCOL（For reference only）
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
Prepare cells in growth medium overnight.
On the next day, add 1X BTC AM working solution into your cell plate.
Note If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
Incubate the dye-loaded plate in a cell incubator at 37 ℃ for 30 to 60 minutes.
Note Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 400/540 nm cutoff 515 nm and Ex/Em2 = 480/540 nm cutoff 515 nm.
