Cell membrane integrity assays

Differential loss in cell membrane integrity between apoptotic and oncotic cells Combination assays Propidium iodide—YO-PRO-1 Acridine Orange-Ethidium Bromide Annexin V-Propidium iodide Useful for live, unfixed cells or unfixed organs only... 

Assays that measure mitochondrial function and cell viability and growth, cell membrane integrity, membrane potential, intracellular ATP, reduced glutathione... 

Altered cell permeability assays The loss of cell membrane integrity can result in the release of certain soluble, cytosolic enzymes Lactate dehydrogenase release assay (tetrazolium salt-based assay) Glucose-6-phosphate dehydrogenase release assay (resazurin-based assay)... 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

Evaluation of membrane integrity is the most commonly used measurement of cell viability, indicating instantaneous or progressive damage over a few hours, and can be performed by 51Cr or specific enzymes release or a dye exclusion assay. These assays are particularly important for toxic agents that exert a primary effect on membrane integrity. Nevertheless, quantification can be difficult due to cell loss by surface detachment or autolysis. 

The pordne proxinud tubule-like cell line LLC-PKi, has been the most widely utilized cell type in the study of the direct tubular effects of CSA. CSA induces direct toxicity to LLC-PKj cells, manifested by an increase in cell vacuolization, a decrease in cell proliferation [171] and a dose dependent decrease in overall cellular viability as measured by Mi l assay and loss of membrane integrity. Sudi gross cell damage, with loss of membrane integrity (increased LDH release and decreases in trypan blue exclusion), is indicative of necrosis [172]. 

The integrity of cell membrane has also to be considered. In that case, another colored assay is performed in the presence of lactate dehydrogenase (LDH). The reaction will occur only if the enzyme is released in the media, implying a leaking of the cell components, thus indicating a degradation of the membrane structure. No effect was detected in the presence of the various Ln helicates complexes. The nontoxicity of the complexes is then proved. 

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