Assays

The methods and the implementation of the assays Calcein AM, Calcium Orange AM and the heartbeat monitoring are displayed here. .

Heartbeat monitoring

The contraction of the heart cells will be monitored by measuring visually the beats per minute of the different heart cell cultures at specific points in time. This needs to be done at 37oC, because the cardiomyocytes will beat slower when they cool down. To make sure the cardiomyocytes do not cool down, a self-made climate chamber will be used to keep the temperature constant. The cells will be monitored real-time using the self-made climate chamber and a microscope with a camera. The acquired footage will be processed using image J. The grey-scale between different cell cultures will be compared.

Calcein assay

To make the cells more visible and to simultaneously measure the percentage of living cells, a viability assay will be used, namely the Calcein assay. Calcein AM is a lipid-soluble substrate which can cross cell membranes. Inside the cell Calcein AM will be converted by the enzyme esterase into a water-soluble fluorescent product which can no longer pass cell membranes freely. Dead cells do not have that enzyme so they won't be coloured.

Calcium orange assay

The calcium flow in and out the cell will be monitored because it provides extra information about whether or not the cells contract in a normal, healthy way. The monitoring of the calcium will be done with Calcium Orange AM. Because of its lipid solubility it can pass through cell membranes freely. Inside the cell esterase enzymes cleave the AM group of the rest of the molecule, revealing its charge. Due to this change in structure it becomes water soluble and can no longer pass the cell membranes. It now stays inside the cell where it binds the Ca2+, which gives a fluorescent colour.