A quantitative method for routine measurement of cell surface P2X7 receptor function in leucocyte subsets by two-colour time-resolved flow cytometry

The P2X(7) receptor is a ligand-gated cation channel activated by extracellular ATP and highly expressed on monocytes, macrophages and lymphocytes. Activation of this receptor by exposure to extracellular ATP opens a selective cation channel that allows Ca2+ and Ba2+ influx, and K+ efflux. Over the first minute the channel adopts a second and larger permeability state allowing the uptake of ethidium(+), followed by a cascade of intracellular downstream effects. Current methods used to study the P2X(7) receptor function, do not give quantitative measurement in sub-populations of a mixed cell suspension. We describe a quantitative method to determine the P2X(7) receptor function using time-resolved two-colour flow cytometry by assessing ATP-induced ethidium(+) uptake. Practical factors such as ethidium bromide concentration, agonists, temperature and buffers are also studied. Moreover, the ATP-induced ethidium(+) uptake method is compared to ATP induced barium (Ba2+) influx with Fura-Red. These two compatible methods can be used to screen the channel/pore function of the cell surface P2X(7) receptor among individuals and the results may be useful to estimate susceptibility of subjects to certain infectious diseases. Crown Copyright (C) 2007 Published by Elsevier B.V. All rights reserved.