title: The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method.
abstract: Most comparative studies on neutrophil (PMN) isolation techniques have shown either activation or functional impairment of the cells due to the different separation processes. We have established a preparation method for PMN from human whole blood employing iron tagged, magnetizable antibodies against the cell surface antigen CD15. The aim of our study was to test whether this magnetic separation (MACS) alters cellular functions of PMN in comparison to a conventional density gradient technique (Percoll). The purity, cell yield, and pre-activation of the cells were evaluated. The latter was assessed by quantifying the expression of the integrin CD11b using flow cytometry. Furthermore, as functional tests, cell morphology and the oxidative burst reaction were investigated. We have shown that the use of 'magnetic' antibodies leads to highly purified PMN (> 99% of isolated leukocytes), while there is still contamination by eosinophils (about 6%) after Percoll separation. Platelet contamination was about the same in both procedures (approx. one platelet per two PMN). The basal expression of CD11b and, hence, neutrophil activation, was significantly lower and the upregulation of CD11b in response to FMLP was more pronounced after magnetic separation, as compared to density gradient centrifugation. The MACS technique did not lead to polarisation of PMN, nor did it affect the oxidative burst. This study suggests that magnetic separation is a simple, time-saving technique, yielding highly purified and functionally intact PMN.
authors.affiliation: Department of Physiology, University of Munich, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Zahler
authors.forename: C
authors.initials: C
authors.lastname: Kowalski
authors.forename: A
authors.initials: A
authors.lastname: Brosig
authors.forename: C
authors.initials: C
authors.lastname: Kupatt
authors.forename: B F
authors.initials: BF
authors.lastname: Becker
authors.forename: E
authors.initials: E
authors.lastname: Gerlach
mesh_terms: D002499:Centrifugation, Density Gradient; D006801:Humans; D018189:Immunomagnetic Separation; D016177:Macrophage-1 Antigen / Q000032:analysis; D009504:Neutrophils / Q000502:physiology*
chemical_list: D016177:Macrophage-1 Antigen