Technical Advance: Autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production

The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.