A Novel Method for Cryopreservation of Human Neutrophils for Phenotypic Analysis

Background: Neutrophils play an essential role in pathogen clearance and can also contribute to pathogenesis and inflammation. Phenotypic changes of neutrophils during infection or inflammation may be associated with their functional activity. However, investigation of neutrophil phenotypes and functions in clinical studies is challenging because of their short viability ex vivo and difficulties in effectively cryopreserving neutrophils for subsequent analyses. Methods: We isolated neutrophils from human peripheral blood and evaluated different approaches for cryopreservation. We examined labelling of cell surface receptors of interest, including CD64, CD32, CD16, CD54, CD66b, CD62L and CD14, on fresh versus cryopreserved neutrophils by flow cytometry. Trypan blue staining was used to assess cell viability and membrane integrity. Results: We developed a neutrophil cryopreservation protocol that preserved cell immunophenotypes for downstream evaluation. All cell surface receptors tested could be labelled and quantified using flow cytometry. There were some differences in levels of surface staining for some receptors when comparing fresh and cryopreserved cells, but these differences did not interfere with the immunophenotypic evaluation of neutrophils. Discussion: Overall, the data suggest that our neutrophil cryopreserving and thawing protocols can preserve neutrophil surface receptors relevant to functions and phenotypes and are methods suitable for use in clinical studies.