A quantitative method for routine measurement of cell surface P2X7 receptor function in leucocyte subsets by...

Area of Science:

• Immunology
• Cell Biology
• Biophysics

Background:

• The P2X(7) receptor, a key component of the innate immune system, is expressed on monocytes, macrophages, and lymphocytes.
• Its activation by extracellular ATP leads to cation channel opening, facilitating Ca(2+) and Ba(2+) influx and K(+) efflux.
• A subsequent larger pore formation allows ethidium(+) uptake, triggering downstream cellular effects.

Purpose of the Study:

• To develop a quantitative method for assessing P2X(7) receptor function in mixed cell populations.
• To optimize parameters for measuring ATP-induced ethidium(+) uptake.
• To compare this method with ATP-induced barium influx for P2X(7) receptor screening.

Main Methods:

• Quantitative measurement of P2X(7) receptor function using time-resolved two-colour flow cytometry.
• Assessment of ATP-induced ethidium(+) uptake.
• Comparison with ATP-induced barium (Ba(2+)) influx using Fura-Red.

Main Results:

• A novel quantitative method for determining P2X(7) receptor function was established using flow cytometry and ethidium(+) uptake.
• Optimization of practical factors like ethidium bromide concentration, agonists, temperature, and buffers was achieved.
• The ethidium(+) uptake method was validated against barium influx, demonstrating compatibility.

Conclusions:

• The developed flow cytometry assay provides a quantitative measure of P2X(7) receptor channel/pore function.
• This method enables screening of P2X(7) receptor function in individuals.
• The results may aid in estimating susceptibility to certain infectious diseases based on P2X(7) receptor activity.