I am not able get any reading from lucigenin (5 microM). What i get is just the background from the samples. The buffer that i use for samples is HEPES. Any suggestions? I have tried using a higher concentration of lucigenin too ..say 20-25 microM. And i ended up reading only the background values. Can anyone let me know what would be the problem? Do we always need to use fresh samples? I used freezed total homogenates from adult rats and also fresh cardiomyocytes.