Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes

Abstract

Quantitative determinations of the cell membrane potential of lymphocytes (Wilson et al., J Cell Physiol 1985;125:72-81) and thymocytes (Krasznai et al., J Photochem Photobiol B 1995;28:93-99) using the anionic dye DiBAC 4 (3) proved that dye depletion in the extracellular medium as a result of cellular uptake can be negligible over a wide range of cell densities. 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-1 and small LCL-HO 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-Hodgkin-Katz 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 4 (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 4 (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. ' 2013 International Society for Advancement of Cytometry Key terms cell density; dye depletion; flow cytometry; membrane potential; mV-scale calibration; oxonol staining IN contrast to electrophysiological methods, the quantitative determination of the plasma membrane potential V by fluorescence measurement using charged dyes requires calibration. If cell subpopulations under study differ in the number of dyebinding sites, they will exhibit an individual relation between fluorescence and V. Consequently, calibration may be necessary more than once within an experiment. Several procedures have been accepted, which, nevertheless, differ considerably in methodological complexity. A direct relation between fluorescence and V can be obtained by the microscopic imaging of cells electrophysiologically clamped at definite voltages (1-4). As this method is quite laborious, the number of measuring values within a reasonable time is quite low. In a lot of studies using spectrofluorimetry (5-13), microscopic imaging (14-23), or flow cytometry (24,25), calibration was usually based on applying a voltage ramp by varying concentrations of extracellular sodium or potassium with isosmolar substitution of the respective...

Complement C5a Alters the Membrane Potential of Neutrophils during Hemorrhagic Shock

“…We established and validated a reliable method for PMN to detect even small changes in transmembrane potential towards hyper- or depolarization ( Figure 1 ), which has been previously described for other cell types and controlled by patch-clamp technique [ 38 , 45 ]. As the slope of the calibration curve remained stable over a certain range of mV, we assume that equilibration of membrane potential to the altered ion concentrations happens at least to a considerable extent as calculated by the GHK equation.…”

“…The dye and/or the time resolution (and the issues discussed below regarding temperature) might be the reason why the results of this work differ quantitatively from other groups [ 9 , 17 , 47 – 49 ], as the reported results here are of lower magnitude. Second, membrane potential is proportionally linked to temperature, which is also represented within the GHK equation [ 37 , 38 ]. While (pre)incubating cells, we stored the specimens at 37°C in a water bath.…”

“…However, as cells are sucked into the flow cytometer, changes in surrounding temperature as well as mixing with rinsing fluid of the system are inevitable. Nonetheless, as temperature and—according to the GHK equation [ 37 , 38 ]—therefore membrane potential are decreased by this effect, we might have rather underestimated instead of overestimated the changes in membrane potential. Third, we cannot measure absolute values of membrane potential, as we have no intracellular detector.…”

“…Isolated PMN were incubated with 40 μ M bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC 4 (3)) for 10 min for measuring the membrane potential. The Goldman-Hodgkin-Katz (GHK) equation [ 37 , 38 ] was used in a simplified version to approximate the cellular membrane potential ( Supplement 1 Equation 1). Intracellular Na + and K + levels were assumed to be 14 mM and 140 mM.…”

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials

“…Some previously cited works (Hezi-Yamit et al 2009;Richards and Ivanisevic 2012) state that increased hydrophilicity disfavors cell attachment on the surfaces. Since the cellular membrane is known to be negatively charged from the outer side at physiological conditions (Emri et al 1998;Klapperstuck et al 2013), phospho-silk fibroin films will be prone to repel seeded cells. Thus, it can be solely stated with a high degree of confidence that lesser attachment will occur.…”