I'm trying to measure intracellular calcium levels in primary-cultured hepatocyte and the protocol was as follows:
1. hypoxia-stimulated cells(in 96wells) were incubated in 5 uM fluo-3/am-containing medium(probenecid was supplemented) for 30 min.
2. wash with PBS for twice
3. Cells were incubated for another 30 min
4. fluorescence was measured at excitation 485 nm, emission 535 nm
After this, Background was abstracted from sample's results
So, my question is,
1. Is this the right protocol? I am so confusing since the data is too fluctuated! TT
2. In some references, cells stained with fluo-3/am was stimulated with hypoxia
hypoxia, staining, which one should be the first?
3. Do somebody have protocols of fluo-3/am staining for confocal/FACS?
Help me TT
I believe that you protocol is accurate. I use a similar protocol, and I have consistent results. I usually add probenecid after the incubation with Fluo-3 as well, to prevent any loss in the staining.
Your protocol sounds fine! I often use fluo-3 am to load cardiomyocytes. 25 min loading with fluo3am, 3-5 micromol at room temperature, then 10 min waiting in normal physiological saline (no fluo) for de-esterification, and then you can measure beautiful Ca signals. Just watch out for your imaging settings (laser power etc). Good luck!
Hi Nari,
i do not which image processing software u use. But be aware that background correction can cause severe differences in the fluorescence intensities of your cells. Furthermore i usually try to wash twice the volume of the cell suspension (e.g. 60µl volume = 2 times 3 x 20 µl steps). Probadly there is some dye left on the well/cell surface which could also influence your signal. Primary system are always more challenging than cell lines. What we realized while working on primary neurons, depending on the feature you want to display you have to analyze the right numbers of cells to overcome and identify sub population and noise effects.
If your interested in some home brewed image analysis plugins or random sampling approaches to define the number of cells you need just send an answer...
Since u did not mention the following points ... I hope you washed the cells again after the last indicator incubation period (twice) - actually one incubation-period in indicator-containing medium of up to one hour should suffice. Secondly, I hope you really used the AM ester version of this dye! Third, cells should rest for at least half an hour in nutrient-rich medium or at least indicator-free buffer in order to allow complete de-esterification. Keeping those points in mind, you could perhaps optimize settings regarding fluorescence on your specific device. If you still observe differences in basal calcium levels, it might be due to varying subsets of different cells in your primary cell-sample.
Have fun while playing around!
I would do Fluo3 staining first than check basal level than do hypoxia and check again for the changes.
Hello Nari
My self not done assay but in future iam planning to do. As per datasheet you need to measure fluorescence at excitation 506nm, emission 526 nm. may be this is reason u may be getting too fluctuated data.
Good evening resepectable everyone,
I need some advice at once very very much
I will try an assay which is about siımultaneous calcium mesaurement by flow cytometry. But there is a problem. I dont have fluo-3 AM but instead I have fluo-3 impermeant potassium salt. I dont have enough time and credit to get fluo-3 AM. and ı have to try that assay. Now, what would you do if you were me ? ı have tried to permeabilise the cells with cytoperm cytofix and I think it did work but ı cant be sure to adjust an appropriate assay protocol. I mean what would you really do to measure the calcium simultaneously with fluo-3 impermeant satl. By the way, I dont have any other techniques like microinjection or scrape loading. I mean the only way is to use fluo-3 salt form. Keep forward to hearing from you honourable ladies and gentlemens.
Best regards
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