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

Abstract

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 Ca(2+) and Ba(2+) 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 (Ba(2+)) 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.