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Fura Red is a visible light-excitable fura-2 analog that offers unique possibilities for ratiometric measurement of calcium ion in single cells by microphotometry, imaging or flow cytometry when used with single excitation, green-fluorescent calcium indicators. Fura Red AM is the cell-permeable version of Fura Red used for noninvasive intracellular loading. Fura Red AM can be simultaneously loaded into cells with Fluo-3 AM, Fluo-8 AM or Cal-520 AM. An advantage of combining two calcium dyes is that dyes with longer excitation wavelengths can be used. This usually causes less harm to the cells than using ratiometric dyes that are excited with UV- or near UV-light (e.g. Fura-2), as light at visible wavelengths is less phototoxic.
Fluorescence microplate reader（for reference only）
Excitation 435, 470
Emission 630, 650
Cutoff Ex/Em = 435/630, cutoff 610. Ex/Em = 470/650, cut off 630
Recommended plate Black wall/clear bottom
Instrument specification(s) Bottom read mode/Programmable liquid handling
Example protocol（For reference only）
Fura Red AM Stock Solution
Prepare a 2 to 5 mM stock solution of Fura Red 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.
Fura Red AM Working Solution
On the day of the experiment, either dissolve Fura Red 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, Fura Red 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 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 Fura Red 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 monitor fluorescence intensity using a fluorescence plate reader, which contains a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 435/630 nm cutoff 610 nm and Ex/Em2 = 470/650 nm cutoff 630 nm.
