Hi!
I’ve been imaging ATP-evoked Ca2+ response from skin fibroblasts. I’ve used 80uM ATP to evoke the response in 0-Ca2+ HBSS. The protocol I used worked fine at first, but when I continued the experiments after a short break, I haven’t been able to get the cells to respond to ATP. On top of the missing ATP response, cells don’t have spontaneous activity at all. When stimulating the cells with the 10uM Ionomycin in the 0-Ca2+ HBSS, the Intensity raises, but relatively little (about 2X baseline values). The cells seem to be healthy and in good condition.
I’m using Fluo-4 AM dye, and I have incubated cells for 50 min RT before imaging.
I can see change in the fluorescent intensity when changing the extracellular media from 1.8M Ca2+ containing HBSS to 0-Ca2+ HBSS, an vice versa.
I have already checked all the reagents and tried to image new cell lines, too, with similar results. The response for the bradykinin is missing, too.
Thank you for the help.
Mrs/miss Rönkkö,
Please consider the following discussion and the reference section therein, together with the sub-references:
[https://www.researchgate.net/post/Beta_value_calculation_of_absolute_intracellular_calcium_concentrations].
In general, the Fs does not appear method of choice for quantitative purposes both in vitro and in vivo. Only the mass spectrometric based analysis can provide reliable analytical information from the perspective of the chemometrics among the currently developed analytical tools and instrumentation.
Dear Julius,
You may already have solved your problem, but if not I thought I'd send a few humble thoughts. Since you've tried new reagents and cells, you have ruled out some of the obvious issues such as the ATP going off or homologous/heterologous desensitisation. However, did you try a new batch of Fluo-4AM? We've had issues where the calcium indicators retain their ability to fluoresce but become less and less responsive to calcium over a few days. This happened even without freeze-thaw cycles (we make a fresh aliquot of calcium indicator for every coverslip of cells that we use, so we don't freeze-thaw). Responses to ionomycin (if it's fresh) are normally pretty reliable in our hands and so can give you an idea of how well the calcium indicator is working.
Your cells do appear to be healthy and nicely fluorescent, but the responses do seem a bit modest (although what you record on the screen will depend on the settings for your imaging system).
One comment- at the start of two of your plots the fluorescence was declining in all the ROIs recorded. Was the decline in the fluorescence due to an experimental manipulation you did just before starting the recording? If not, the drop in fluorescence would be something I'd personally need to understand. I always like to start experiments with a perfectly level baseline value for a few minutes (which is straightforward in most cells if there isn't background spontaneous calcium signalling going on). If the decline in fluorescence wasn't due to an experimental manipulation then the Fluo4 fluorescence is being lost somehow. Are you using laser light to illuminate your cells on a confocal microscope, or are you using a wide-field system? Just wondering if there is some photobleaching occurring (can happen easily with laser illumination). How long do you leave your cells after loading with Fluo4 to allow for full de-esterification of the indicator?
Apologies if this is of little use. It's sometimes hard to diagnose issues from afar.
Best of success with your experiments.
Martin
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