You need to load the cells with Fura-2 10 miroM, and use epifluorescence with a double exitation of 340/380 nm, then calculate the ratio between the emision signal.
Thanks for yours answers.
@Luis Gonano : 10 µM is high, for cells in suspension I saw 3-5 µM, is it logical ? what is the incubation time with Fura-2 10 µM for adherent cells ? I saw for cells in suspension : 30 minutes at 37 °C and after washing, 15 minutes at room temperature for de-esterification of Fura-2. Is that enough to get a signal epifluorescence in adherent cells?
thank you !
It depends on your sample, the excitation light intensity and the quality of your lens and aqusition camera. I would start using 5 microM and 60-80 min of incubation. In standard equipment you will have enough light. If not, increase the incubation time but not fura2 concentration.
Oh sorry! I used 10 microM but after change to 5 microM loading during 12 minutes in fresh cardiac myocytes at room temperature, with 30 minutes after loading to ensure de-esterification.
Ok Carlos & Luis. Thanks for this details, I will test this differents conditions it's a good starting point, thanks a lot it's help me ! :-)
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