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CAS:145037-81-6
Storage:Powder:-20℃，2 years;Insolvent(Mother Liquid):-20℃，6 months;-80℃，1 year
Purity:≥95%
Appearance:Solid
Calcium ion fluorescence probe Rhod-2, AM is a reagent for calcium flux determination, which is the preferred method in drug discovery for screening G-protein-coupled receptors (GPCR).
Rhod-2 is a Ca2+ fluorescence probe with high affinity to visible light excitation wavelength. Rhod-2 AM is an acetyl methyl derivative of Rhod-2 and has cell membrane permeability. Once inside the cell, it is cleaved by its lactase enzyme to produce membraneless permeable Rhod-2, which remains in the cell to perform its corresponding physiological functions. Maximum excitation/emission wavelength: 549/578 nm.
Fluorescence microscope（for reference only）
Excitation TRITC filter set
Emission TRITC filter set
Recommended plate Black wall/clear bottom
Fluorescence microplate reader（for reference only）
Excitation 540
Emission 590
Cutoff 570
Recommended plate Black wall/clear bottom
Instrument specification(s) Bottom read mode/Programmable liquid handling
Example protocol（For reference only）
Rhod-2 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Rhod-2 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.
Rhod-2 AM Working Solution
For most cell lines, Rhod-2 AM at a final concentration of 4-5 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 Rhod-2 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 Rhod-2 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 TRITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.
