Response to genistein: Assaying the activation status and chemotaxis efficacy of isolated neutrophils

Neutrophil (PMN) activation and chemotaxis toward inflammatory stimuli play critical roles in host defense and tissue inflammation. To determine the molecular mechanisms that regulate PMN function, many studies currently employ in vitro PMN activation and transmigration assays using freshly isolated peripheral PMN or PMN isolated from bone marrow. However, due to the highly sensitive nature of PMN, cell activation or priming can occur during isolation, which demands assay(s) that ensure the consistency of isolated PMN prior to using them in subsequent experiments. Here, we introduce a simple screening assay based on the observation that in transmigration assays, isolated PMN differentially respond to the tyrosine kinase inhibitor genistein and this is related to their activation status. As shown, we observed that isolated PMN for which early migration is enhanced by genistein have an overall high transmigration efficacy and that over 80% of applied PMN migrate across collagen-coated filters in a 2 h time period. Conversely, the inhibitory/non-enhancement effect of genistein is accompanied by a poor PMN transmigration, with less than 25% of applied PMN transmigrating across. Further analysis of PMN spontaneous adhesion, degranulation and cell surface CD11b/CD18 expression suggests that reduced migration of PMN is associated with PMN activation/priming that happens, in most cases, during the in vitro cell isolation procedure regardless of the blood donor. Thus, based on these observations, we developed a “genistein assay” to directly predict PMN status after each isolation. From our experience, this assay has not only revealed new insights into the mechanisms of PMN activation and assisted in functional assays, but it has also provided a method that can be mastered by both inexperienced and experienced researchers to assay isolated PMN and thus avoid using inconsistent cells (e.g. pre-activated PMN) in their experiments.