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

The P2X7 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 P2X7 receptor function, do not give quantitative measurement in sub-populations of a mixed cell suspension. We describe a quantitative method to determine the P2X7 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 P2X7 receptor among individuals and the results may be useful to estimate susceptibility of subjects to certain infectious diseases.