Biochemical and Physiological Alterations in Canine Neutrophils Separated by Lysis or Percoll Gradient Isolation Technologies

Abstract

Neutrophils were isolated from the peripheral blood of clinically normal canines by hypotonic lysis or density gradient (Percoll) centrifugation techniques. As a function of preparative technique, separated neutrophils were examined in vitro for alterations in membrane lipid integrity, and both granular and cytosolic specific enzymes. Membrane lipid disorder was assessed by merocyanine 540 (MC540), a fluorescent bioprobe, which intercalates into disrupted cellular lipid membranes. Evidence of membrane lipid disorder was based upon comparisons of mean cellular fluorescence exhibited by unstimulated cells. Based upon comparisons of mean cellular fluorescence the isolation methodologies did not appear significantly different. Significant levels of membrane disruption, due to preparative technique, were evident upon neutrophil stimulation with phorbol myristate acetate (PMA). Neutrophils which were Percoll-separated and PMA-stimulated demonstrated significant levels of membrane disruption not apparent in lysis-separated stimulated aliquots of cells. Cellular isolation methods did not significantly alter cellular myeloperoxidase (MPO) levels based on comparisons of cellular totals. Extracellular lactate dehydrogenase (LDH) levels of lysis-separated cells were four-times those of Percoll-separated neutrophils.