Calcium Fluorescent Probe Fluo-8 AM£¬IC-1578

Introduction

Calcium measurements are critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy, and fluorescence microplate readers. Fluo-3 AM and Fluo-4 AM are most commonly used among the visible light-excitable calcium indicators for live-cell calcium imaging. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8 dyes are developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of Ex/Em = ∼490/∼520 nm. Fluo-8 AM can be loaded into cells at room temperature, while Fluo-3 AM and Fluo-4 AM require 37¡ãC for cell loading. In addition, Fluo-8 AM is two times brighter than Fluo-4 AM and four times brighter than Fluo-3 AM.

Product Size

250ug  1mg

Example protocol

1.PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 ¡ãC after preparation. Avoid repeated freeze-thaw cycles

2.Fluo-8 AM Stock Solution

Prepare a 2 to 5 mM stock solution of Fluo-8 AM in high-quality, anhydrous DMSO.

3.PREPARATION OF WORKING SOLUTION

Fluo-8 AM Working Solution

3.1 On the day of the experiment, either dissolve Fluo-8 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

3.2 Prepare a 2 to 20 µM Fluo-8 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, Fluo-8 AM at a final concentration of 4-5 ¦ÌM 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 Fluo-8 AM. A variety of Pluronic F-127 solutions can be purchased from InCellGene .

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 InCellGene .

4.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.

4.1 Prepare cells in growth medium overnight.

4.2 On the next day, add 1X Fluo-8 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.

4.3 Incubate the dye-loaded plate in a cell incubator at 37 ¡ãC for 30 to 60 minutes.

Note: Incubating the dye for longer than 2 hours can improve signal intensities in certain cell lines.

4.4 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.

4.5 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 490/525 nm cutoff 515 nm.
Safety Information

Please wear gloves, lab coat and goggles while operating. Prevent contact product directly. In case of contacting, wash with large amount of water.
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