Flow cytometric kinetic assay of the activity of Na⁺/H⁺ antiporter in mammalian cells

Abstract

Background:The Na ϩ /H ϩ exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H ϩ ion in exchange for extracellular Na ϩ and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. Methods: To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate.

Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma

“…We used a kinetic flow cytometry assay in which recovery from intracellular acidification induced by propionic acid is dependent on NHE activity and is measured as ΔpH s −1 (ref. 40). Treatment with MEK inhibitor or digitoxin alone did not significantly affect NHE activity in either melanoma (Fig.…”

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation

“…Besides analysis of baseline and activation end points, the introduction of the "Time" parameter in the early 1980s (22) allowed to measure changes of cellular parameters over time (23). Since then, publications using kinetic flow-cytometry started to be reported (24)(25)(26)(27)(28). Subsequently, the technique was improved and redefined as "continuous" (29) or "real-time flow cytometry" (30,31).…”

A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals

“…Determination of NHE activity.-For assaying NHE activity, a new flow cytometric kinetic assay was followed as described early (8). Briefly, cells were prepared as indicated above for determination of pHi.…”

“…The time-course of fluorescence ratio was plotted from Listmode files using WinMDI 2.8 public domain software and the rate of recovery of pHi was calculated using curve-fitting routines with Sigma Plot 8.02 program. The activity of NHE was defined as the initial recovery rate of pHi after acidification (8).…”