Nuclear factor activation by FcgammaR in human peripheral blood neutrophils detected by a novel flow cytometry-based...

In mammals, neutrophils are the most abundant circulating leukocytes. Neutrophils are short-lived cells presenting at least two important transcriptionally regulated cellular responses, initiated by cell activation: the production of pro-inflammatory cytokines and the inhibition of apoptosis. The study of transcriptionally regulated processes in these cells cannot be approached through conventional reporter gene strategies, as there are currently not available methods for neutrophil transfection. Here we describe a novel flow cytometry-based method that allowed quantification of nuclear factor NF-kappaB activation in neutrophils, in response to FcgammaIIA and FcgammaRIIIB stimulation. The sensitivity of this method allowed the detection of small changes in NF-kappaB activation, due to pharmacological inhibition of receptor-initiated signaling pathways. NF-kappaB activation was also detected by this method in various leukocyte cell lines. In addition, quantification of Fcgamma receptor-initiated nuclear activation of ERK and Elk-1 was successfully achieved through this method. The broad applicability and versatility of this flow cytometry-based method position it as a fast and reliable alternative to traditional methods for analyzing activation of transcription factors in a variety of cell types.