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Human neutrophil isolation and cell culture

SK Sarah E. Kleinstein
JM Jamison McCorrison
AA Alaa Ahmed
HH Hatice Hasturk
TD Thomas E. Van Dyke
MF Marcelo Freire
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Isolation of human neutrophils from whole blood was conducted by Ficoll-Histopaque density-gradient centrifugation (Histopaque-1077 and Histopaque-1119; Sigma-Aldrich), as has been described previously [12]. Briefly, neutrophils were isolated after isotonic lysis of red blood cells and counted with a hemocytometer; neutrophil viability (> 95%) was assessed using trypan blue live/dead staining [51]. Monocyte exclusion was completed by plating. A separate monocyte fraction was isolated for monocyte counting. For culture experiments, isolated neutrophils were incubated with RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS (v/v) (Life Technologies) at 37 °C. Wright-Giemsa staining was used to identify individual cell types and confirm neutrophil isolation and purity (routinely > 98%) [12].

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