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 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.
Jursik
Claudia
C
Department of Medicine, the University of Sydney, Nepean Hospital, Penrith, New South Wales 2750, Australia.
Sluyter
Ronald
R
Georgiou
Jennifer G
JG
Fuller
Stephen J
SJ
Wiley
James S
JS
Gu
Ben J
BJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D001464:Barium / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000378:metabolism / Q000493:pharmacokinetics; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015203:Reproducibility of Results; D012964:Sodium / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D013696:Temperature
D002712:Chlorides; D004791:Enzyme Inhibitors; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D001464:Barium; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D012964:Sodium; D004996:Ethidium; D011188:Potassium; D002118:Calcium