Hello Sara, I have worked on Fura-2 for my project. This dye is ratiometric and your fluorescence microscope must have the illumination system for 340 nm and 380 nm. We set the microscope protocol in such a way that both 340 and 380 nm wavelengths are excited at each time point (alternative 340 and 380 nm). The ratios of these two intensities are then calculated in an excel sheet to get the final value of intensities.

• DOI: 10.1152/ajplung.00206.2019

Hello Sangeeta Bhallamudi,

Thank you for your reply and for sharing your paper.

I have had a few tries with the system in my group to acquire the fluorescence pictures. At the moment, I am just unsure whether what I am doing during data analysis is what it is supposed to be done.

Thank you for your help.

Sara, the first question is when you say analyze calcium data using pluripotent cells, what exactly is your experiment? Do you expect the calcium levels to change after you stimulate the cells with an agonist/antagonist?

To put it simply, you load your cells on a coverslip, treat with Fura2, put the coverslip on an imaging chamber, and hit the cells with alternating wavelengths of 340nm and 380nm. Fura2 is a ratiometric dye, meaning the same dye shows different absorption maxima depending on if it bound to calcium or not. Free Fura2 shows a maximum absorption at 380nm compared to bound Fura2 which shows at 340nm. You plot a graph with Time on X axis and ratio of Fura2 RFU at 340nm/RFU at 380nm. Based on the treatment, if your treatment affects calcium in the cells, you should see a change in the ratio of Fura2 after the treatment. Hope this helps. Feel free to ask any questions you have.

Best,

Goutham