Calcium measurement is 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. Rhod-FF AM is cell-permeable, and generates Rhod-FF upon esterase hydrolysis in cells. Rhod-FF has a lower binding affinity for Ca²⁺ and is suitable for Ca²⁺ measurements from 10 to 200 μM. Like the parent Rhod-2 indicator, Rhod-FF is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca²⁺.
Product Advantages
1. Low background fluorescence: Fluorescence signal is greatly enhanced after binding to Ca²⁺, which effectively reduces background interference and improves detection sensitivity.
2. Wide application range: It can monitor dynamic changes of intracellular free Ca²⁺ concentration via fluorescence microscopy, flow cytometry, fluorescence spectroscopy, fluorescence microplate readers and other instruments.
3. Low calcium binding affinity: Suitable for calcium ion detection within the concentration range of 10–200 μM.
Application Scope
For intracellular calcium ion measurement.
KEY PARAMETERS
Fluorescence microscope
Emission: TRITC filter set
Excitation: TRITC filter set
Recommended plate: Black wall/clear bottom
Fluorescence microplate reader
Cutoff: 570
Emission: 590
Excitation: 540
Recommended plate: Black wall/clear bottom
Instrument specification(s): Bottom read mode/Programmable liquid handling
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Rhod-FF AM to given concentration.
Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg |
|---|
| 1 mM | 87.268 μL | 436.338 μL | 872.676 μL | 4.363 mL | 8.727 mL | | 5 mM | 17.454 μL | 87.268 μL | 174.535 μL | 872.676 μL | 1.745 mL | | 10 mM | 8.727 μL | 43.634 μL | 87.268 μL | 436.338 μL | 872.676 μL |
|
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
Rhod-FF AM Stock Solution
Prepare a 2 to 5 mM stock solution of Rhod-FF AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
Rhod-FF AM Working Solution
1. On the day of the experiment, either dissolve Rhod-FF AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
2. Prepare a 2 to 20 μM Rhod-FF 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, Rhod-FF 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-FF AM. A variety of Pluronic® F-127 solutions can be purchased from Aladdin.
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 Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from Aladdin.
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.
1. Prepare cells in growth medium overnight.
2. On the next day, add 1X Rhod-FF 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.
3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note: Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
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.
5. 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.
