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.

Methods of Granulocyte Isolation from Human Blood and Labeling with Multimodal Superparamagnetic Iron Oxide Nanoparticles

“…In addition, there is no consensus on the best technique for isolating granulocytes to achieve high purity. The methods applied for granulocyte isolation are diverse [29][30][31][33][34][35]53,[67][68][69][70], and their efficiency has been reported in different ways, such as cellular viability or cell isolation yield, cell functionality, number of target cells and contaminants after isolation, which makes it difficult to evaluate the methods for clinical application.…”

“…In this method, the Percoll gradients were constructed using different densities by diluting Percoll with PBS (Gibco ® , Carlsbad, CA, USA). Gradient solution 3 (G3, Figure 6) and gradient solution 4 (G4, Figure 6) contained 60% and 70% Percoll, respectively, as described in previous studies [35,70,105]. The Percoll solutions with PBS were obtained by slowly adding the following sequence: 3 mL of gradient 4 (G4), 3 mL of gradient 3 (G3) and 3 mL of the leukocyte mixture gently deposited on top, taking care to avoid mixing.…”

“…The most commonly used method uses gradients of Percoll [29][30][31][32][33][34]. Isolation purity, viability and yield are important factors for evaluating each method and ensuring cellular integrity [29,[34][35][36].The noninvasive evaluation of acute inflammatory processes makes it possible to analyze molecular and cellular processes in vivo, usually based on targeting specialized cells such as macrophages, lymphocytes or granulocytes to the desired target [37]. Noninvasive imaging techniques that provide structural, functional and molecular information in preclinical and clinical studies include CT, MRI, positron emission tomography (PET), single-photon emission computed tomography (SPETC) and near-infrared fluorescence (NIRF) imaging, among others [38][39][40], each of which has advantages and limitations.…”

“…The most commonly used method uses gradients of Percoll [29][30][31][32][33][34]. Isolation purity, viability and yield are important factors for evaluating each method and ensuring cellular integrity [29,[34][35][36].…”

Procedures for leukocytes isolation from lymphoid tissues and consequences on immune endpoints used to evaluate fish immune status: A case study on roach (Rutilus rutilus)

“…Among the different techniques, hypertonic lysis and density gradient are the two main way used in literature to separate leukocytes from erythrocytes. Many isolation methods of vertebrates leucocytes exists and were extensively studied as hypertonic shock using ammonium chloride [14], positive selection [15], self-generating gradient [16,17], cryopreservation [18], adherence and agglutination [19] and density gradient. The use of Ficoll® or Percoll® to constitute density gradients is one of the most common isolation procedure for vertebrate leucocytes [20].…”