title: Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods.
abstract: Studies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.
authors.affiliation: a Department of Pathological Sciences , Santa Casa de São Paulo School of Medical Sciences , São Paulo , Brazil.
authors.forename: Tainá
authors.initials: T
authors.lastname: Mosca
authors.affiliation: a Department of Pathological Sciences , Santa Casa de São Paulo School of Medical Sciences , São Paulo , Brazil.
authors.forename: Wilma C N
authors.initials: WC
authors.lastname: Forte
mesh_terms: D000328:Adult; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D002634:Chemotaxis, Leukocyte; D064368:Healthy Volunteers; D006801:Humans; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism*; D010587:Phagocytosis; D055815:Young Adult
keywords: Cell separation; chemotaxis; methods; neutrophils; phagocytosis