%0 Journal Article
%T Calcium Transient Assays For Compound Screening With Human Ipsc-derived Cardiomyocytes: Evaluating New Tools - Calcium Transient Assays For Compound Screening With Human Ipsc-derived Cardiomyocytes: Evaluating New Tools - Open Access Pub
%A Joseph Vecchi
%A Neil J. Daily
%A Pinar Kemanli
%A Radleigh Santos
%A Tetsuro Wakatsuki
%J OAP | Home | Journal of Evolving Stem Cell Research | Open Access Pub
%D 2017
%X Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes. DOI10.14302/issn.2574-4372.jesr-16-1395 Fluorescent dye indicators for intracellular calcium measurement are widely used in many fields of biology. The fluorescent calcium indicator dyes work by their ability to change their brightness when bound to calcium ions. Fura-2 was one of the first calcium indicators developed and has a large spectral shift upon binding to calcium that allows for the ability to ratiometrically measure intracellular calcium concentration 1. Newer calcium indicators have minimal spectral shift upon calcium binding but are much brighter and are used for measuring qualitative changes in calcium concentration. FLIPR dyes were developed as a no-wash calcium dye to minimize the number of steps and washing artifacts important for high throughput applications 2, 3. FLIPR dyes come with background quenching technology which may improve SNRs and decrease standard deviation and dye loading does not require detergent. Cal-520 AM is the latest generation of fluorescence indicators in the line of Fluo3-AM and Fluo4-AM which are dyes linked to non-polar acetoxymethyl (AM) esters that allow for efficient crossing of the cell membrane and are rapidly hydrolyzed by esterases inside the cell. AM conjugated dyes are reconstituted in DMSO and may be used with a low concentration detergent, such as 0.04% Pluronic F-127, to increase dye loading,
%U https://www.openaccesspub.org/jesr/article/414