An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.
A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37 degrees C followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 +/- 4% at 89 +/- 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml. Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 microM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.
Koenderman
L
L
Central Laboratory, Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Kok
P T
PT
Hamelink
M L
ML
Verhoeven
A J
AJ
Bruijnzeel
P L
PL
D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002634:Chemotaxis, Leukocyte; D004804:Eosinophils / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D010972:Platelet Activating Factor / Q000494:pharmacology; D013189:SRS-A / Q000096:biosynthesis; D013997:Time Factors
D010972:Platelet Activating Factor; D013189:SRS-A; D009240:N-Formylmethionine Leucyl-Phenylalanine