Browsing by Author "Bowen, John M."

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    Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry
    (Cytotechnology, 1997) Kisaalita, William S.; Bowen, John M.
    With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (Vm) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by Vm establishment. The (Vm)-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 104 cells/cm2. At higher initial cell densities, under differentiating culture conditions, Vm development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development.
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    Effect of Medium Serum Concentration on N1E-115 Neuroblastoma Membrane Potential Development
    (Animal, 1997) Kisaalita, William S.; Bowen, John M.
    Propidium iodide (PI) emissions were determined by PMT2 after pas-sage through a 610-nm long-pass filter. SSC was determined by PMT4. FALS signals were linearly amplified with a gain of 2. Unless otherwise stated, 50,000 events were counted at an approximate rate of 200 events per second. Because subcellular debris has low FALS, this parameter was used to gate out these particles. Oxonol fluorescence analysis was restricted to events that were PI-negative, since dead or dying cells are stained by PI. To compare results for experiments conducted at different times, polystyrene fluorospheres (Coul-ter Corporation, Hialeah, FL) were used to set the PMT,(oxonol signal) and PMT4 (SSC signal) at channel numbers of 35? 1 and 100? 2, respectively, before each experiment. DMSO was included in this study as a positive control, because it is considered one of the most potent enhancers of electrical excit-ability among other chemical differentiating agents (Spector and Baumgold, 1982). Aminopterin was also included as a positive con-trol. Aminopterin blocks the normal biosynthetic pathway by which nucleotides are made (Henderson et al., 1965) and therefore was expected to kill off most if not all the dividing (nondifferentiating) cells and thus yield the most differentiated cells. Previous Vm results obtained by intracellular and patch clamp recording techniques on a few cells have indicated that the transition from low to high Vm occurred between 7 and 10 days (Kimhi et al., 1976; Santone et al., 1986; Baumgold and Spector, 1987; Cosgrove and Cobbett, 1991).