I have used fura-2 to demonstrated changes in cytoplasmic calcium in chondrogenic cells. Article Adrenocorticotropin Evokes Transient Elevations in Intracell...

When loading with fura-2 you may want to keep the cells at room temperature. When the cells are at 37 degrees they tend to take the dye into the ER where calcium is stored. Many protocols recommend 37 degrees but form my experience room temperature works better. Additionally, use 340/380 as your excitation wavelengths and 510 as your emission wavelength. 360 is the isobestic point for fura-2 and this may be why your ratio is off.

It is expected that the RFU's would increase after adding ionophore because you have opened all the cellular calcium stores. www.jbc.org/content/262/26/12801.full.pdf

Are you measuring basal levels or are you measuring cytoplasmic calcium flux in response to an activator?

Dear Amanda Gross,

make sure you are using the right filters. As Jodi F Evans already pointed out: 340/380 would be the optimal excitation and 510 nm the emission wavelength.

You worked at the isosbestic point (which is actually not as fix as we would love it to be), we would expect Ca2+ independent fluorescence. However, you observed a huge increase in RFU that is unexpected.

I think it is possible that you used the wrong emission filter.

Best regards

Hi Jodi and David,

Thanks for your helpful comments. As Jodi suggested, I tried the experiment where cells were incubated with Fura-2 AM dye in room temperature vs. in 37 degree incubator. I believe in previous years our lab had used the 335/505 and 363/512--I'm not sure why, but it's used in other papers as well--maybe to lessen background noise?

Indeed, using the 335/363 ratio appeared to give an expected ratio of ~1.0 at baseline unstimulated, along with the previously mentioned unexpected ratio of ~0.6 when using the ionophore.

However, when I measured using the 340/380 ratio, baseline control ratio was higher than 2.0. Also, using 340/380 did not change the issue of ionophore giving lower than control ratio (still at about 0.6).

Incubating at RT vs. 37 degrees did not seem to really impact loading or change the unstimulated and/or ionophore-stimulated ratios observed at either 335/363 or 340/380, so I did not bother labeling the various lines in the attached images.

I'm stumped! Thanks again in advance.

Hi Amanda,

the signal should not get brighter in both channels if you not change the dye concentration. As you mentioned all the filters are set correctly. I never used two different emission wavelength. However, they are close together so this shouldn't have a huge impact...

Are you sure that the device works correct? Can you test it with an established Fura2-based assay?

Is there a volume change in one of the groups?/ do you use a solvent control?

Can you check with a microscope if there are Fura2 crystals in your cells suspension? It might be possible that they get dilute if you add a DMSO diluted agent. This might change the dye concentration.

Also check if the compound is fluorescent itself... Just add it without cells.

Best,

David

Yes I agree with David try using another agent to elevate calcium levels. A23187 (Calcimycin) has an intrinsic fluorescence excitable by UV light, making it less useful with UV-excitable Ca2+ indicators, e.g., Fura-2, but it is still useful for long-wavelength Ca2+ indicators, e.g., Fluo-2. In my studies I have used digitonin for calibration.

hi, Amanda！I'm doing the same work and meet the same question, but I use another cell, HEK293T, have you solved this problems? would you share same tips?

thank you a lot!