A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils.
The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.
Menegazzi
R
R
Istituto di Patologia Generale, Università di Trieste, Italy.
Zabucchi
G
G
Knowles
A
A
Cramer
R
R
Patriarca
P
P
D000001:Calcimycin / Q000494:pharmacology; D002469:Cell Separation; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000097:blood; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology; D009195:Peroxidase / Q000032:analysis / Q000097:blood*; D010544:Peroxidases / Q000032:analysis / Q000097:blood*; D010972:Platelet Activating Factor / Q000494:pharmacology; D012016:Reference Values; D013053:Spectrophotometry / Q000379:methods; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D007202:Indicators and Reagents; D009842:Oligopeptides; D010972:Platelet Activating Factor; D000001:Calcimycin; C024998:N-formylnorleucyl-leucyl-4-phenylalanine; D015054:Zymosan; D007770:L-Lactate Dehydrogenase; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase; D013755:Tetradecanoylphorbol Acetate