Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability 1,2 . The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells 3 . PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5 . The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6 . In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases 7,8 , allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9 . Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment 10 . Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence 10 . Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).

A great deal of variability exists in American local government structures. It would be useful to analyze the differences in outcomes associated with different structures, but two factors stand in the way. First, there is no agreement about how to classify city governments. Second, many of the components used in existing classifications are inaccurately reported or missing in survey data. In this research note, a new classification system based on three easily determined components is developed and used to measure the current distribution of forms and variations in an expanded data set that includes almost all cities over 10,000 in population. Keywords form of government, council-manager, mayor-councilIn local government, Americans can choose among a wide range of structural features that determine form, methods of election, and assignments of responsibilities

Th e standard narrative to explain the evolution of roles fi lled by city managers suggests that managers originally served as administrative technicians who carried out the policy directives of city councils, but, over time, they became more involved in policy advice and community leadership. Th is article documents enduring involvement in policy and the recent expansion of community leadership, analyzing defi nitions of the manager's roles off ered by practitioners and scholars since the beginning of the city manager profession 100 years ago. In addition, measurements of the manager's role performance at various points in time are examined, along with trends in the preparation of managers and the context in which they work. Ignoring the evidence that the policy role is integral to professional management can lead to unfounded questions about the legitimacy of city and county managers and impairs our understanding of the council-manager form of government and political-administrative relations.

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