Neutrophil Isolation from Nonhuman Species

Abstract

The development of new advances in the understanding of neutrophil biochemistry requires effective procedures for isolating purified neutrophil populations. Although methods for human neutrophil isolation are now standard, similar procedures for isolating neutrophils from many of the nonhuman species used to model human diseases are not as well developed. Since neutrophils are reactive cells, the method of isolation is extremely important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animals (bovine, equine, ovine), small animals (murine and rabbit), and nonhuman primates (cynomolgus macaques), and describe optimized details for obtaining the highest cell purity, yield, and viability. We also describe methods to verify phagocytic capacity in the purified cell populations using a flow cytometry-based phagocytosis assay.

Neutrophil transcriptional profile changes during transit from bone marrow to sites of inflammation

“…15,[42][43][44] The neutrophil literature contains numerous methodologies for neutrophil purification; ranging from density gradient centrifugation to immunomagnetic selection, cell sorting and culture models. 14,41,[45][46][47] In this study, obtaining high-quality mRNA from neutrophils for all three compartments was the greatest obstacle and required the most amount of refining. While the current optimal method for obtaining pure neutrophils is immunomagnetic negative selection, 15,43,48 this approach resulted in very low cell yields (loss of up to 90% of neutrophils-see the section on 'Materials and methods') and low mRNA quality.…”

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

“…Mouse bone-marrow cells were isolated as described before (36). Erythrocytes were lysed with ACK lysis buffer for 3 min at room temperature.…”

Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

“…Purification of murine PMNs. Murine PMNs were isolated from pooled bone marrow from 8-to 12-week-old RML Swiss-Webster mice as described previously (15,22). Bone marrow was flushed from the femurs and tibias of 3 or 4 RML mice and pooled.…”

“…Following Percoll and Histopaque 1119 density gradient purification, PMNs were resuspended in RPMI medium and enumerated by hemocytometer counts. PMN viability was 95 to 99% as determined by trypan blue dye exclusion (22). PMN purity was estimated to be 70 to 85% by hemocytometer direct counts and by white blood cell differential counts.…”