Abstract
Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.
Highlights
That were shown to influence neutrophil recovery and response to s timulation[27]
We determine the individual effects of delayed sample processing, staining temperature, and type of anticoagulant employed on neutrophil phenotype by directly comparing eight common methods of neutrophil characterization in relation to neutrophil recovery, activation, and multiplet formation
As a reference for method comparison, the “whole blood diluted” method (M1; Fig. 1a) was utilized. This method is based on a rapid dilution of a small amount of stained whole blood in DPBS at 1:2000 final ratio immediately prior to sample analysis by flow cytometry with acoustic focusing technology without further processing
Summary
That were shown to influence neutrophil recovery and response to s timulation[27]. Current knowledge regarding the effects of these factors on neutrophil phenotype is limited. We determine the individual effects of delayed sample processing, staining temperature, and type of anticoagulant employed on neutrophil phenotype by directly comparing eight common methods of neutrophil characterization in relation to neutrophil recovery, activation, and multiplet formation. Our data indicate that staining whole blood at 4 °C within 3 h of blood draw, removal of unbound antibodies to minimize nonspecific antibody binding, followed by treatment with One-step Fixation and RBC Lysis buffer (hereafter referred to as Fix/lyse buffer) minimizes neutrophil activation and multiplet formation while maximizing neutrophil recovery
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