HelloI am measuring the calcium concentration in cancer cells...

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Sarbari Acharya
Oct 30, 2014
protocol Protocol: Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry
Hello

I am measuring the calcium concentration in cancer cells using Fura-2Am. I am unable to understand the Rmin and Rmax values needed to be taken for calibration of the experiment. I am using EGTA (3 uM) and Digitonin (100 um) for RMax and R min respectively. But I am not get
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Sarbari Acharya Answered Nov 6, 2014

Institute of Life Sciences

Hi

And what about incubation time of EGTA or Cacl2
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María Fernández-Velasco Author Answered Nov 5, 2014

Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hi
You should first incubated cells with FURA and then with EGTA.
Incubation with FURA is 30-40 min.
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Sarbari Acharya Answered Nov 5, 2014

Institute of Life Sciences

Hi

Thanks for the suggestion. Could you tell me what is the incubation time for Rmin and Rmax. Whether the cells should be simultaneously incubated with both EGTA (3 uM) and Fura-2am at the same time. Or first should i pre-incubate cell with EGTA followed by Fura2am loading.
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María Fernández-Velasco Author Answered Nov 3, 2014

Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hello you should calculated R Min and Rmax separately
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Sarbari Acharya Answered Oct 30, 2014

Institute of Life Sciences

Hello

Thanks for your suggestion. But I just wanted to know that whether Rmin and Rmax will be calculated separately or in the same system
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María Fernández-Velasco Author Answered Oct 30, 2014

Cell Communiation, Instituto de Investigaciones Biomédicas Alberto Sols, Spain

Hello,
maybe you can try to calibrate with 2.5 mM CaCl2 (Rmax) and 10 mM EGTA (Rmin). Morover there are some comercial kits to make a calibration curve (check in Invitrogen/Molecular Probes)
María/Lisardo
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