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In contrast, most flow cytometric studies have not verified this condition but rather assumed it from the start. Consequently, the initially prepared extracellular dye concentration has usually been used for the calculation of the Nernst potential of the dye. In this study, however, external dye depletion could be observed in both large IGR\u20101 and small LCL\u2010HO cells under experimental conditions, which have often been applied routinely in spectrofluorimetry and flow cytometry. The maximum cell density at which dye depletion could be virtually avoided was dependent on cell size and membrane potential and definitely needed to be taken into account to ensure reliable results. In addition, accepted calibration procedures based on the partition of sodium and potassium (Goldman\u2010Hodgkin\u2010Katz equation) or potassium alone (Nernst equation) were performed by flow cytometry on cell suspensions with an appropriately low cell density. The observed extensive lack of concordance between the correspondingly calculated membrane potential and the equilibrium potential of DiBAC<jats:sub>4<\/jats:sub>(3) revealed that these methods require the additional measurement of cation parameters (membrane permeability and\/or intracellular concentration). In contrast, due to the linear relation between fluorescence and low DiBAC<jats:sub>4<\/jats:sub>(3) concentrations, the Nernst potential of the dye for totally depolarized cells can be reliably used for calibration with an essentially lower effort and expense. \u00a9 2013 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.22300","type":"journal-article","created":{"date-parts":[[2013,5,6]],"date-time":"2013-05-06T15:47:16Z","timestamp":1367855236000},"page":"612-626","source":"Crossref","is-referenced-by-count":13,"title":["Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes"],"prefix":"10.1002","volume":"83A","author":[{"given":"Thomas","family":"Klapperst\u00fcck","sequence":"first","affiliation":[{"name":"Department of Dermatology and Venereology Martin Luther University of Halle\u2010Wittenberg Halle (Saale) 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