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

J Immunol Methods. 2007 Aug 31;325(1-2):67-77. doi: 10.1016/j.jim.2007.06.002. Epub 2007 Jun 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology
  • Adenosine Triphosphate / pharmacology
  • B-Lymphocytes / drug effects
  • B-Lymphocytes / metabolism
  • Barium / metabolism
  • Biological Transport / drug effects
  • Calcium / pharmacology
  • Chlorides / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Ethidium / metabolism
  • Ethidium / pharmacokinetics
  • Flow Cytometry / methods*
  • Humans
  • Killer Cells, Natural / drug effects
  • Killer Cells, Natural / metabolism
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism*
  • Monocytes / drug effects
  • Monocytes / metabolism
  • Potassium / pharmacology
  • Receptors, Purinergic P2 / metabolism
  • Receptors, Purinergic P2 / physiology*
  • Receptors, Purinergic P2X7
  • Reproducibility of Results
  • Sodium / pharmacology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism
  • Temperature

Substances

  • Chlorides
  • Enzyme Inhibitors
  • P2RX7 protein, human
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X7
  • Barium
  • KN 62
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • Adenosine Triphosphate
  • Sodium
  • Ethidium
  • Potassium
  • Calcium