Calcium fluorescence assays

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

Selected entries from Methods in Enzymology [vol, page(s)] Chelation, 238, 74, 76, 297 buffers [for analysis of exocytosis, 221, 132 preparation, 219, 186 modulation of cytosolic buffering capacity with quin2, 221, 159] fluorescence assay, 240, 724-725, 740-742 fluorescence imaging, 225, 531 238, 303-304, 322-325, 334-335 free intracellular levels after bacterial invasion, 236, 482-489 free calcium in solutions for membrane fusion analysis, calculation and control, 221, 149 homeostasis mechanisms, 238, 80 hormonal elevation, 238, 79 inositol phosphate effect on release, 238, 207 determination of cytosolic levels [computer methods, 238, 73-75 with fura-2, 238, 73, 146 with indo-1, 238, 298, 316-317 with quin-2, 238, 297] hormone effects, 238, 79 ionomycin effects, 238, 79 membrane depolarization effects,... 

Fig. 7 Biosynthesis of NATs and TRP channel activation by NATs. (a) Evidence for a fatty acyl CoA taurine A-acyltransferase activity was detected in mouse tissue by incubating taurine and arachidonoyl-CoA with various tissue lysates, (b) arachidonyl NAT was tested as an activator of the TRPV1 (black line), TRPV4 (gray line), and TRPM8 (dashed line) ion channels. Channel activation was measured using a Fura-2-based calcium-imaging assay, where the ratio between the fluorescence at 340 and 380 nm is reflective of cellular calcium concentrations...

Liu, E. C. K. Abell, L. M. Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader. Anal. Biochem. 2006, 357, 216-224. 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Fluorimetric methods have proven useful for the assay of metal ions in solution 1 e.g., in vivo studies of calcium-selective fluorescence probes have been reported by... 

The choice of the mobile phase is very important, as fluorescence is sensitive to fluorescence quenchers. Highly polar solvents, buffers, and halide ions quench fluorescence. The pH of the mobile phase is also important to fluorescence efficiency for example, quinine and quinidine only display fluorescence in strongly acidic conditions, whereas oxybarbiturates are only fluorescent in a strongly alkaline solution [67,68]. Due to the stability of the chromatographic sorbents, the use of very acidic or basic mobile phase may not be possible. One alternative is to alter the effluent pH postcolumn. Postcolumn addition of sulfuric acid has been used for the assay of ethynodiol diacetate and mestranol in tablets [69]. Another example is the determination of tetracycline antibiotics in capsules and syrup where EDTA and calcium chloride were added to enhance fluorescence [70]. 

Measure the fluorescence emission at 505 nm using 340 nm excitation. Monitor the baseline. When a stable baseline is obtained for 10-30 s, add the chemokine to be tested. After the peak of calcium mobilization has subsided (about 1 min), calibrate the assay by adding 20 pL of minimum buffer, allow for stabilization, then add 20 pL of maximum buffer to dissolve cell membranes in order to obtain full complexation of fura-2 with Ca2+. Record the baseline, the peak, the minimum and the maximum values (see Fig. 1). The concentration of Ca2+ mobilized is calculated according to the following modified formula (6) ... 

High throughput flash luminescence readers such as the FDS6000 and 7000, as well as the Lumax Flash HT, enable functional GPCR and calcium channel testing. A sensitive photon-counting CCD camera enables aequorin and luciferase activity to be measured by flash luminescence. Thus, these advances allow fluorescence-based assays to be replaced by luminescence-based assays,... 

CFDA (Sigma) is used at 40 )Ug/ml final concentration in serum free medium, 0.1% BSA, pH 6.0, for 30 min at 37°C. At the end of the assay, adherent labeled tumor cells are placed in 0.2% SDS for 30 min at 37°C to release the fluorescent marker. Three volumes of calcium-magnesium-free-PBS are added, and the fluorescence of the cell lysate is measured with a Perkin-Elmer LS-5 luminescence spectrophotometer (excitation maximum 485 nm, emission maximum 538 nm) (Price et al., 1995). 

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