Membrane Potential and Changes in Ion Permeability

The generation of intermediate oxygen radicals after monocyte and neutrophil stimulation is accompanied by changes in membrane potential. Lipophilic dyes such as the cyanine compounds dipentyloxacarbocyanine (DiOC5[3]) and dipropyl-thiocarbocyanine (DiSC5[3]) can be used to measure this aspect of cellular activation. These dyes diffuse into the cell with different localization patterns depending on their concentration. Cellular activation is followed by a loss of

Fig. 3. Flow cytometric enumeration of (left panel) cells in control wells (no HUVECs) and (right panel) cells that have migrated through wells containing HUVECs. The number of events counted in 60 s was recorded. The control count represents the total possible number of migrated cells. The actual number of migrated cells is represented as a percentage of the total possible number.

Fig. 3. Flow cytometric enumeration of (left panel) cells in control wells (no HUVECs) and (right panel) cells that have migrated through wells containing HUVECs. The number of events counted in 60 s was recorded. The control count represents the total possible number of migrated cells. The actual number of migrated cells is represented as a percentage of the total possible number.

cell-associated fluorescence, which is related to changes in membrane potential. Stimulation with PMA results in an irreversible loss of fluorescence caused by an irreversible membrane depolarization; however, stimulation with the chemotactic agents fMLP or C5a results in an initial loss of fluorescence with a nadir at approx 2 min, followed by a gradual return to the baseline level. A dose-dependent reduction in fluorescence during the initial phases of stimulation can be observed with increasing concentrations of the chemotactic stimuli. Due to the heterogeneity of the response to fMLP, a small proportion of neutrophils do not respond or do not show the biphasic response to the stimulus when this peptide is used. These potentiometric cyanine dyes have been shown to be independent of the action of oxygen radicals and of mitochondrial activity, although they accumulate into these organelles.

The activation of phagocytic cells leads to an initial cytoplasmic acidification because of the generation of large amounts of protons by NADPH oxidase. As a consequence, proton-conducting channels and the Na2+-K+ antiport are activated, leading to sustained intracellular acidification. These two aspects of intracellular pH changes may be measured by flow cytometry. Intracellular changes of pH in response to either soluble or particulate stimuli may be monitored by means of the fluorochrome carboxy-seminapthorhodafluor-1-acetomethyl ester (SNARF-1/AM) (45). This dye, which is modified by elastase, is loaded into the cell and fluoresces in the orange region of the light spectrum. However, with increasing pH, the fluorescence moves to the red region of the spectrum. A ratio of the red/orange fluorescence is calculated through the calibration of cells with high K+ buffers of defined pH in the presence of nigericin, a polyether ionophore that carries monovalent cations across membranes with high specificity for K+. A 575- to 595-nm band-pass filter and a 620-nm long-pass filter have to be used to evaluate acidic orange SNARF-1/AM and basic SNARF-1, respectively.

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