We are working on chimeric receptors, which are CCR5 and CCR7. After transfection and the cell line expressed the receptors, we tried to investigate the calcium influx within the cell, with or without application of stimulation. We have already got some data when using the living cell specimens to observe the calcium influx, together with Fluo-4 AM. However, since the image resolution of our reserve fluoroscent microscope is not that nice, I am trying to do the coverslips. Therefore, dose anyone have any experience, to say whether the fixation processes by 4% PFA may have the effects on the Fluo-4 AM dye within the cells?
Many thanks.
Are you hoping to observe fluorescence caused by intracellular Ca2+ in a fixed cell population on a coverslip? Fixation will prevent your receptors from working naturally, and indeed prevent the dye from responding to Ca2+. Plus, the cells will be dead, and the membrane permeated.
What information are you hoping to gain by doing this?
Thanks Paul. What I concern is whether the PFA fixation process may affect the fluoroscence of Fluo-4 AM. Since we stimulate the living cells and apply Fluo-4 AM as the suggesting protocol, to observe the intracellular Ca2+. However we don't know, for instance, the PFA fixation processes may cause the permeablisation of the cell membrane, and sequentially cause the leakage of the intracellular Ca2+, results in the attenuation of the fluoroscence. Paul G. Morris
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