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Use of Fluo-8® AM Esters

1. Load Cells with Fluo-8® AM Esters:

AM esters are the non-polar esters that readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. AM esters are widely used for loading a variety of polar fluorescent probes into live cell non-invasively. However, cautions must be excised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted just before use in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions may be stored desiccated at –20 °C and protected from light. Under these conditions, AM esters should be stable for several months.

Following is our recommended protocol for loading Fluo-8® AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.

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

b)       On the day of the experiment, either dissolve Fluo-8® in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a working solution of 1 to 10 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.02% Pluronic® F-127. For most of cell lines, Fluo-8® reagents with a concentration ranging from 4-5 uM are recommended. The exact concentration of the indicator required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.

Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Fluo-8® AM esters.  A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.

c)       If your cells containing the organic anion-transports, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators.

Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest.

d)       Add equal volume of the dye working solution (from Step b or c) into your cell plate.

e)       Incubate the dye-loading plate at a cell incubator or room temperature for 20 minutes to one hour.

Note: Decreasing the loading temperature might reduce the compartmentalization of the indictor.

f)        Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 2.5 mM probenecid, if applicable) to remove excess probes.

g)       Run the experiments at Ex/Em  = 490/525 nm

Use of Screen Quest™ Fluo-8 NW Calcium Assay Kits for HTS Applications

GPCR activation can be detected by direct measurement of the receptor mediated cAMP accumulation, or changes in intracellular Ca²⁺ concentration. GPCR targets that couple via Gq produce an increase in intracellular Ca²⁺ that can be measured using a combination of Fluo-8® reagents and a fluorescence microplate reader. The fluorescence imaging plate readers (such as, FLIPR™, FDSS or BMG NovoStar™) have a cooled CCD camera imaging system which collects the signal from each well of a microplate (both 96 and 384-well) simultaneously. These plate readers can read at sub-second intervals, which enables the kinetics of the response to be captured, and has an integrated pipettor that may be programmed for successive liquid additions. Besides their robust applications for GPCR targets, our Screen Quest™ Fluo-8 Calcium Assay Kits can be also used for characterizing calcium ion channels and screening calcium ion channel-targeted compounds.

Figure 2. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of the Screen Quest™ Fluo 8-NW calcium assay kit and Fluo-4 NW kit (according to the manufacturer’s instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG LabTech) to achieve the final indicated concentrations. The EC₅₀ of Fluo-8 NW is about 1.2 uM.

Compared to other commercial calcium assay kits that either based on Fluo-3 or Fluo-4, our Screen Quest™ Calcium Assay Kits have the following advantages for HTS applications:

•Broad Applications: work with both GPCR and calcium channel targets.

•Convenient Spectral Wavelengths: maximum excitation @ ~490 nm; maximum emission @ ~514 nm.

•Flexible Dye Loading: dye loading at room temperature (rather than 37 ºC required for Fluo-4 AM).

•No Wash Required and No Quencher Interference with Your Targets.

•Robust Performance: enable calcium assays that are impossible with Fluo-4 AM or Fluo-3 AM.

•Strongest Signal Intensity: 2 times brighter than that of Fluo-4 AM; 4 times brighter than that of Fluo-3 AM.

Use of Fluo-8® Salts

Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in fluorescence microplate readers) of the indicators in solutions with precisely known free Ca²⁺ concentrations. Calibration solutions can be used based on 30 mM MOPS EGTA Ca²⁺ buffer. In general, water contains trace amount of calcium ion. It is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca²⁺ concentrations

A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA

B. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl₂

To determine either the free calcium concentration of a solution or the K_d of a single-wavelength calcium indicator, the following equation is used:

[Ca]_free = K_d[F ─ F_min]/F_max ─ F]

Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, F_min is the fluorescence intensity in the absence of calcium and F_max is the fluorescence intensity of the calcium-saturated probe.

The dissociation constant (K_d) is a measure of the affinity of the probe for calcium. The calcium-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ response calibrations of intracellular indicators typically yield K_d values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca²⁺ buffers in the presence of ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin or Triton® X-100 can be used to expose the indicator to the controlled Ca²⁺ levels of the extracellular medium. The K_d values of Fluo-8® reagents are listed in Table 1 for your reference.

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