Ca2+ is a universal second messenger that can influence many aspects of cellular pathophysiology.
Unlike cardiomyocytes and neuronal cells, the study of Ca2+ handling in cardiac
fibroblasts (CF) is challenging, since these cells are thin, sensitive and quit heterogeneous, i.e.
individual CF may respond to varying degrees upon stimulation, and that requires the use of a
large number of cells to get reliable data. This protocol describes a high throughput method for
real-time monitoring and semi-quantification of Ca2+ handling in primary cultured rat and
human cardiac fibroblasts under basal conditions and following stimulation with Ang II. The
assay is based on the utilization of “Screen Quest Fluo8-No Wash Kit” from AAT Bioquest.
The kit utilizes Fluo-8 AM Ca2+ sensitive fluorescent dye, which is more than 2 times brighter
than Fluo-4 AM, and 4 times brighter than Fluo-3 AM, according to the manufacturer. The
manufacturer mainly designed the kit to be easily adopted for automated monitoring of total
change of fluorescence intensity in cells cultured in 96-or 384-well plate. However, this method
does not allow determination of parameters for individual cells, monitoring individual cell status
and reactions to the treatment conditions; therefore we present here a modified protocol that
deals with these limitations.
Keywords: 3-6 words. Cardiac fibroblasts, live cell calcium imaging, calcium transient,