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This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample. The fluorescent dyes used in this protocol include 5(6)-carboxyfluorescein diacetate (CFDA), which indicates the functional activity of esterase inside viable bacterial cells, and DRAQ7, a dye that exploits membrane-compromised bacterial cells to enter and stain the cell. The live cell population stained with CFDA emits a fluorescent green color while the dead cell population stained with DRAQ7 emits a fluorescent red color, which allows the two populations to be distinctively separated by the IFCM system. Additionally, the cytometer captures a clear image of each object, which can then be analyzed for morphology features. The IFCM system is able to reliably, accurately, and precisely determine a bacterial cell concentration as long as the concentration of cells in a sample is no less than 1 × 10 3 cells/ml. The two dyes, CFDA and DRAQ7, have been demonstrated to be an effective stain combination for bacterial viability analysis.

Alzheimer's disease is characterized by extracellular plaques that cause neurodegeneration. The major constituent of the plaque is the fibrillar form of the amyloid β (Aβ) peptides of 39–43 amino acids long. Physiological levels of copper and zinc ions have been shown to cause marked aggregation of Aβ peptides. Spectroscopic studies have identified metal ion coordination sites on Aβ peptides, binding affinity and stoichiometry, although discrepancies exist among various studies. To complement spectroscopic approaches and to obtain binding energetics, we have used isothermal titration calorimetry to examine the interaction of copper and zinc ions with Aβ‐(1–16), the N‐terminal portion of the longer Aβ peptide that is known to contain all the coordination sites for the two ions. Titration orders of metal ion into peptide and peptide into metal ion were carried out at pH 5.5 and 6.5 at several temperatures. Two stoichiometries were obtained for each ion depending on the injection order: two metal ions bound per peptide when the peptide was titrated into metal ion and one metal ion bound per peptide when metal ion was titrated into peptide. Analysis of the result suggests that two different binding modes were employed by each ion depending on the injection order. Copper binds more strongly than zinc. For both ions, binding was enthalpy driven; affinity increases with increasing pH, suggesting deprotonation coupled to binding, a result consistent with histidine participates in metal coordination. It was also found that prolonged incubation of the peptide with copper or zinc induces an additional slow process that is best explained by a conformational change, possibly, aggregation of Aβ‐(1–16) which in turn increased the affinity of the peptide for the two metal ions.

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