Research paper Nuclear factor activation by FcγR in human peripheral blood neutrophils detected by a novel flow cytometry-based method Erick García-García, Carlos Rosales ⁎ Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico Received 29 June 2006; received in revised form 15 September 2006; accepted 15 December 2006 Available online 16 January 2007 Abstract 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-κB activation in neutrophils, in response to FcγIIA and FcγRIIIB stimulation. The sensitivity of this method allowed the detection of small changes in NF-κB activation, due to pharmacological inhibition of receptor-initiated signaling pathways. NF-κB activation was also detected by this method in various leukocyte cell lines. In addition, quantification of Fcγ 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. © 2006 Elsevier B.V. All rights reserved. Keywords: FcγRIIA; FcγRIIIB; Fc receptor; NF-κB; ERK; Elk-1 1. Introduction In mammals neutrophils are the most abundant cir- culating leukocytes (Huizinga et al., 1990; Sendo et al., 1996). At the onset of an inflammatory process neutrophils are rapidly recruited to sites of infection, where they act as the first line of defense against in- vading pathogens (Becker, 1990; Sklar and Omann, 1990; Johnson et al., 1992). In contrast to other leu- kocytes, such as lymphocytes or macrophages, neu- trophils are short-lived cells that undergo apoptosis, unless activated through various membrane receptors during the inflammatory process (Sendo et al., 1996; Simon, 2003). Neutrophil activation is also required for the initiation of the various defense mechanisms displayed by neutrophils, which include phagocytosis, respiratory burst, release of various microbicidal molecules by degranulation, and production of pro- inflammatory cytokines (Downey et al., 1995; Scapini et al., 2000; Burg and Pillinger, 2001). Though neutrophils are characterized by having large stores of molecules involved in defense mechanisms (Faurschou Journal of Immunological Methods 320 (2007) 104 – 118 www.elsevier.com/locate/jim ⁎ Corresponding author. Department of Immunology, Instituto de Investigaciones Biomédicas - UNAM, Apdo. Postal 70228, Cd. Universitaria, México D.F. - 04510, Mexico. Tel.: +52 55 5622 3883; fax: +52 55 5622 3369. E-mail address: carosal@servidor.unam.mx (C. Rosales). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.12.006