|Shipping||Standard overnight for United States, inquire for international|
|Dissociation constant (Kd, nM)||320|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Minimize light exposure|
Dissociation constant (Kd, nM)
|Recommended plate||Black wall/clear bottom|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Bottom read mode/Programmable liquid handling|
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Prepare a 2 to 5 mM stock solution of Cal-520® AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-520® AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Cal-520® AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Cal-520® AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Cal-520® 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 Cal-520® AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
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. A variety of ReadiUse™ Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.
SAMPLE EXPERIMENTAL PROTOCOL
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 Cal-520® AM working solution to 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 °C for 1 to 2 hours.
Note: Incubating the dye for longer than 2 hours 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 an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
Common stock solution preparation
|0.1 mg||0.5 mg||1 mg||5 mg||10 mg|
|1 mM||90.666 µL||453.33 µL||906.659 µL||4.533 mL||9.067 mL|
|5 mM||18.133 µL||90.666 µL||181.332 µL||906.659 µL||1.813 mL|
|10 mM||9.067 µL||45.333 µL||90.666 µL||453.33 µL||906.659 µL|
|Mass (Calculate)||Molecular weight||Volume (Calculate)||Concentration (Calculate)||Moles|
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