The major drawback of using Fluo-4 AM is that it requires an organic anion transporter inhibitor, such as probenecid, to prevent leakage. This can hinder the studies that require extended monitoring time and longer cellular retention. To address the issue, a novel Ca2+ indicator, Calbryte 520 AM, was developed. We compared the performance of Fluo-4 AM and Calbryte 520 AM following prolonged incubation periods after cell loading. Cells loaded with Calbryte 520 AM retained the dye for up to 24 h while exhibiting significant fluorescence brightness and superior Fmax/F0 ratios (Fmax: fluorescence intensity upon stimulation; F0: intensity before stimulation). It demonstrated that the longer retention of Calbryte 520 AM can be exploited to accommodate for the extended time required when monitoring calcium dynamics.
Keywords: Calbryte 520 AM; calcium indicator; fluorescence microscope; fluorescence plate reader; probenecid free.