Histochem Cell Biol (2008) 130:281–297 1994; Kheradmand et al. 1998; Kindzelskii and Petty 2002). Indeed, oscillations in the respiratory burst have been cor- related with ﬂuctuations in neutrophil cell shape (Wymann et al. 1989). Petty and colleagues (Petty and Kindzelskii 2000; Kindzelskii and Petty 2002; Petty 2004, 2006) have advocated the use of high-speed imaging to study metabolic waves and cellular signaling in living neutrophils. Neutro- phils were monitored during the process of cell adhesion to a substratum and it was shown that there were waves of NAD(P)H autoﬂuorescence and cytosolic pH changes associated with attachment (Petty and Kindzelskii 2000). They did not monitor whether ROS production occurred during attachment in that study. However, in polarized neutrophils (crawling cells) NAD(P)H waves were associ- ated with production of ROS (Kindzelskii and Petty 2002). These authors stated: ‘‘We have previously noted the pres- ence of longitudinal NAD(P)H waves in elongated neutrophils (Petty et al. 2000). Thus, cell shape (spherical vs. elongated) correlates with the dissipative metabolic patterns observed (circular vs. longitudinal)’’ (Petty and Kindzelskii 2000). These observations emphasize further the need to compare cells activated in suspension and those activated attached to a surface and to study the role of cell shape in neutrophil function. In cytochemical preparations carried out with neutro- phils in suspension, intracellular sites of ROS production have been observed (Kobayashi et al. 1998; Brown et al. 2003). We have shown, as have others, that the intra- cellular trafﬁcking of membrane-bounded compartments in activated neutrophils can be very dramatic and complex (Kobayashi and Robinson, 1991; Robinson et al. 1999). This point is illustrated further herein (see Fig. 7). There- fore caution should be used when comparing neutrophils (and other cells as well) activated in suspension to those activated when attached to a substratum. Membrane traf- ﬁcking events occurring with cells in suspension may not be exactly replicated with attached cells and vice versa. It would be useful for this issue to be addressed in a formal manner such that the intracellular trafﬁcking patterns for membrane compartments of individual neutrophils acti- vated in suspension or when attached to a substratum could be determined and compared. Reactive oxygen species in other cell types While this review has focused narrowly on phagocytic leukocytes and neutrophils in particular, it must be men- tioned that ROS have assumed considerable importance beyond phagocytes. Indeed, ROS production is wide spread throughout the animal and plant kingdoms. The literature on this topic has grown considerably over the past 10 years or so and illustrates the pervasiveness of ROS production in biological systems and the importance of ROS in various 293 biological functions. A review of this area, no matter how cursory, is well beyond the scope of this paper. However, there are numerous excellent review articles available on these topics (e.g., Geiszt and Leto 2004; Lambeth 2004; Terada 2006; Bedard and Krause 2007; Bedard et al. 2007; Lambeth 2007; Brandes and Schro¨ der 2008). Acknowledgments The author is indebted to Drs. John A. Badwey, his post doctoral colleague, and Morris J. Karnovsky, his post doc- toral mentor, for many fruitful discussions about neutrophils and macrophages and their biochemical and cell biological properties. The late Dr. Manfred L. Karnovsky also added greatly to our dis- cussions. I am also grateful to Dr. Richard T. Briggs for generously sharing his knowledge of neutrophils and enzyme cytochemistry when I was new to phagocyte biology. All of these interactions began at Harvard Medical School. At Ohio State University, I was privi- leged to begin collaborations with Drs. Toshihiro Kobayashi and Toshihiro Takizawa that have continued after they returned to Japan. Dr. Harumichi Seguchi has also contributed to our understanding of neutrophils through the use of cytochemistry. The bacterial minicells shown in Fig. 4 were generously supplied by Dr. Bernard M. Babior. I also wish to thank my current colleague Dr. William E. Ackerman IV for assistance in preparing Fig. 1. References Abo A, Webb MR, Grogan A, Segal AW (1994) Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. 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