The quenching effect was especially severe at excitation wavelengths 345 nm to 355 nm, and was still appreciable at 360 nm. However, it was essentially eliminated at excitation wavelengths greater than 364 nm. As the excitation wavelength increased, the fluorescence of the product predictably decreased. At an excitation wavelength of 368 nm, however, AMC fluorescence was decreased by only 40%.
Recommended instrument settings, choice of microplates, and estimated assay sensitivity
Based on the results shown in Figure 3, 368 nm was selected as the excitation wavelength. The next step was to select the emission wavelength and cutoff filter. To help in the selection process, (signal-background)/background1/2 was plotted versus wavelength. The plots were prepared from scans with no emission cutoff filters, with a 420 nm cutoff filter and a 435 nm cutoff filter (Figure 4.) The optimum emission wavelength appeared to be approximately 467 nm. The cutoff filters gave comparable results in that region and both were somewhat better than no cutoff filter. This was confirmed experimentally by running the enzyme assay and blanks at different emission wavelengths with multiple replicates and estimating limits of quantitation. Based on the results above, we recommend that Z-DEVD-AMC-based caspase-3 assays be run on the SPECTRAmax GEMINI with excitation/emission wavelengths set to 368/467 nm with a 435 nm or a 420 nm emission cutoff filter.
CHOICE OF MICROPLATES
White plates give a higher fluorescent signal (and background) than do black plates because the light is reflected, rather than absorbed. (The