Hello,

Concerning the part of Fura 2-AM versus time, it is because you are supposed to follow the response of your cells on a tested time scale in response to a certain treatment (based on your experiment/aim).

Basically, Fura2 is not your treatment, it is your probe to mark calcium (the time and concentration may vary between cell lines, so you have to optimize your experimental setup).

Hence, you don't care about the response per time of Fura incubation (per se) but rather post your treatment of interest.

Concerning Fura incubation, it can be done by both HBSS or medium. For me I didn't notice any difference, however, I prefer to incubate with the cell line medium to avoid any biased results or alterations that could happen on the cells' behavior.

If by analysis you mean "during Calcium Imaging", you have definitely to use HBSS rather than cells' culture medium because phenol red will give signal background and it will be a mess.

I hope I answered your questions

Good Luck

Hi,

Fura-2 is a ratiometric calcium indicator dye which allows us to measure accurate intracellular calcium concentration. If you want to monitor the uptake of extracellular calcium, you should add calcium to your HBSS buffer. Further restrictions on experimental setup arises depending on whether you want to monitor the uptake by intracellular stores such as endoplasmic reticulum (use blocker thapsigargin) or mitochondria (use blocker ruthenium red).

I have used Perkin Elmer spectrofluorimeter which has an in-built software to measure exact concentration of calcium in your experimental setup.

Thanks,

Alpana

Fura-2 and Fura-2 AM is a ratiometric dye that is excited at 340 and 380 nm which is advantageous since it always emits at 510 nm. The reason it is advantageous is that your getting two fluorescent measures of the fluctuation of intracellular calcium which then you can take the emission output and graph it as a ratio to decrease confounding effects such as unequal loading of the cells, differences in cell thickness, and when you are calibrating in order to "approximate" intracellular Ca2+, which I honestly used different kits, cells, made my own Ca2+ concentration gradients, and then would try to calculate [Ca2+] utilizing Rmin and Rmax using the equation from the manuscript below:

Article Fura-2 measurement of cytosolic free Ca2+ in monolayers and ...

[Ca intracellular] = Kd X (R - Rmin)/(Rmax - R) * Sb2/Sbf

Even with using 0 Ca 2+ 5mM EGTA to get my baseline from minutes and using up to in 50 mM caffeine puffs to elicit sharp releases from the SR to obtain a Rmax which was an instanteous release i was never able to really obtain a true intracellular Ca2+ level and this was my major project. I found it to be much more accurate to keep you regions of interest to be consistent size of the recording window, number of cells, loading time (incubation time), wash time, and create chamber capable of making multiple rapid bath changes or run continuous flow, and lastly to keep the level of the fluid in the chamber consistent from recording to recording.

After I mastered these things, my recordings became very reproducible.

I would suggest reading the manuscript above of the mechanics of how the dye works. It really helped me troubleshoot and set up my own flow system which was key to doing more complicated experiments.