Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining.

Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells. Key features Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy. Protocol can be applied to any type of cell that is adherent in culture, including primary cells. Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes. Graphical overview Imaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy. Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).

In vitro Assay to Evaluate Cation Transport of Ionophores.

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions. This protocol was validated in: Proteomics (2022), DOI: 10.1002/pmic.202200061 Graphical abstract.