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,

angiotensin II

Conference Title

INTERNATIONAL EURASIAN CONFERENCE ON BIOLOGICAL AND CHEMICAL SCIENCES

Conference Country

Turkey

Conference Date

April 26, 2018 - April 27, 2018

Conference Sponsor

Interlab

Additional Info

Conference Website