Granulocyte chemotaxis measured in a Boyden chamber assay by quantification of neutrophil elastase.
The human polymorphonuclear leukocyte reaction in response to chemotactic or chemokinetic stimulation is often assayed using the Boyden chamber technique. We present a quick and reliable method for evaluating Boyden chamber experiments, which avoids time-consuming cell counting and does not require expensive equipment. This method is based on assaying human neutrophil elastase, a serine protease derived from polymorphonuclear leukocytes. We tested the method in different types of Boyden chambers equipped with two superimposed filters or a filter amnion membrane combination. The chambers were incubated with the cells for 2 h then dismantled and the elastase activity in supernatant, filters or membrane was assayed. The results were compared with the results obtained by cell counting, or measured by determination of myeloperoxidase. There was a good correlation between the cell count and elastase technique (r = 0.90), but the elastase method achieved higher intra- and interassay precision. Myeloperoxidase and elastase results also correlated well (r = 0.94) and showed comparable intra- and inter-assay precision. With the elastase method it was also possible to quantify polymorphonuclear leukocyte reactions on an amnion membrane surface. In amnion membrane assays the percentage of cells which reacted in response to formyl-peptide stimulation was not altered by varied cell concentrations, and polymorphonuclear leukocytes showed little unstimulated adherence or migration.
Pelz
G
G
Lehrstuhl für Biochemie, Fakultät für Chemie, Universität Bielefeld, Germany.
Schettler
A
A
Tschesche
H
H
D000650:Amnion; D002634:Chemotaxis, Leukocyte / Q000502:physiology; D006098:Granulocytes / Q000502:physiology; D006801:Humans; D007958:Leukocyte Count; D008722:Methods; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000032:analysis*; D009195:Peroxidase / Q000032:analysis
D009195:Peroxidase; D010196:Pancreatic Elastase