Propidium iodine (PI, Sigma, Shanghai, China) staining was used to monitor cell membrane integrity.
The finding that SREBP processing is controlled by different lipids in mammals and flies (sterols and phosphatidylethanolamine, respectively) suggests that an essential function of SREBP is to monitor cell membrane composition and to adjust lipid synthesis accordingly.
To monitor cell membrane permeabilization we expressed the fluorophore CFP together with HKx-YFP.
Additionally, given that one essential function of SREBP appears to be its ability to monitor cell membrane composition and to adjust lipid synthesis accordingly [56], it is possible that the interaction of macrolides with the membrane provides a strong signal to the cell to compensate for membrane disruptions.
Cell permeability to propidium iodide was used to monitor cell membrane integrity as an indicator of apoptosis.
PI (5 ng/ml) was added to medium for monitoring cell-membrane integrity.
We monitored whole cell membrane capacitance transients (Cm) induced by the activation of Cav1.2 subunits [α11.2 (dN60-del1773), α2δ and β2A] without and with SNAP-25, Sx 1A and synaptotagmin (SytI) (excitosome complex) expressed in Xenopus oocytes, as previously described [29] (Fig. 1A).
In the first assay, cell subsets were identified using fluorescently labeled antibodies, and viability was determined by staining with 50 μg/mL of propidium iodide (PI) to monitor losses in cell membrane integrity.
For example, GFP has been used for protein localization (Valadi et al. [2004]), intracellular pH measurement (Valkonen et al. [2013]), for monitoring cell growth mode and cell membrane robustness (Carlquist et al. [2012]).
Thus, monitoring ATPase activity in cell membrane preparations or in purified membrane proteins allows for identification of those compounds that interact with the drug efflux transporters.
