290 these cells were attached to plastic surfaces coated with proteins (e.g., serum, ﬁbronectin), tumor necrosis factors induced massive release of ROS. It was also observed in that study that neutrophils attached to surfaces in the absence of tumor necrosis factors also produced low levels of ROS. Another study of adherent neutrophils also found low levels of ROS release in the absence of additional stimuli (Suchard and Boxer 1994). It is interesting to note that rabbit synovial ﬁbroblasts that had undergone integrin- mediated cell shape change had activated Rac1 which was correlated with the generation of ROS by these cells (Kheradmand et al. 1998). As already noted above, Rac is a component of the assembled NADPH oxidase. Taking all of these factors into account, studies directed toward understanding, in a detailed manner, how shape change itself plays a role in generation of ROS and how shape change modulates ROS production in response to stimu- lation in phagocytic leukocytes seem in order. Exogenous ﬂuorescence probes used for monitoring ROS production have been reviewed (Gomes et al. 2005). Exogenous ﬂuorescent probes have been used in several studies of phagocytic leukocytes. The utility and the limi- tations of current exogenous ﬂuorescent probes for use with phagocytes has been reviewed (Yeung et al. 2005). Important considerations for use of the exogenous ﬂuo- rescent probes, such as dihydroethidine and dihydrorhdamine 123 include mode of entry into cells and the retention of the ﬂuorescent product at the site of ROS generation as well as the linearity of the response. A few examples of the use of exogenous ﬂuorescence probes with neutrophils will be presented. Kobayashi et al. (2000) have used ﬂuorescent probes for ROS detection [succinimidyl ester of dichlorodihydroﬂuorescein diacetate (H2DCFDA)] and endocytosis [TRITC-dextran] to monitor intracellular trafﬁcking in phorbol myristate acetate-activated neutro- phils. They found intracellular sites of ROS production and a complex pattern of the interaction of the ROS-positive compartments and endocytic vesicles. A reduction in H2DCFDA was noted in the presence of fungal gliotoxin (Li et al. 2004). Streptolysin-O permeabilized neutrophils were tested for production of ROS using H2DCFDA. In this study, the ROS-induced ﬂuorescence was monitored by ﬂow cytometry and ﬂuorescence microscopy (Brown et al. 2003). It was found that ﬂuorescence was associated with intracellular structures with a granule-like labeling pattern in the permeabilized neutrophils. An alternative strategy to applying the ﬂuorescent probes directly to cells consists of covalent attachment of the ﬂuorescent probe to phagocytizable particles. In this way, the ROS-induced changes in ﬂuorescence will be restricted to the phagosome (Yeung et al. 2005). These same authors discuss the potential of using ROS-sensitive ﬂuorescent proteins to monitor ROS production. These proteins could 123 Histochem Cell Biol (2008) 130:281–297 be expressed in phagocytic cells, in some cases, or in the target microorganism. This is an attractive prospect, how- ever, the harsh conditions encountered in the phagosome should be taken into account in the design of environmen- tally-sensitive ﬂuorescent proteins. Expression of ﬂuorescent proteins in primary phagocytes, such as human neutrophils, is problematic so the use of microorganisms expressing the ROS-sensitive ﬂuorescent protein as targets for phagocytosis would be preferable in this instance. Fluorescence microscopy has been used in conjunction with a micromanipulator in order to move phagocytic tar- gets to human neutrophils, thus allowing the study of changes in cytosolic Ca2+ and the respiratory burst asso- ciated with a single phagocytic event at a deﬁned site in individual cells (Dewitt et al. 2003). The ﬂuorescent probes Fura-2 and dichlorodihydroﬂuorescein were used to mon- itor changes in cytosolic Ca2+ and production of ROS, respectively. Two Ca2+ peaks were observed to be asso- ciated with phagocytosis but only the second peak correlated with initiation of the respiratory burst. An enzyme cytochemical experiment is shown to illus- trate the dramatic nature of the morphological alterations neutrophils can display during activation. Cytochalasin B, a compound that disrupts ﬁlamentous actin in cells, has been widely used in conjunction with stimuli such as f- Met-Leu-Phe to enhance the release of ROS and the exo- cytosis of intracellular granules (e.g., Zurier et al. 1973; Honeycutt and Niedel 1986; Mukherjee et al. 1994). It has also been shown that cytochalasin B reduces the cortical tension and cytoplasmic viscosity in neutrophils (Tsai et al. 1994). We have studied the effects of cytochalasin B and f-Met-Leu-Phe stimulation on the morphology of human neutrophils. Cerium-based cytochemistry for the detection of alkaline phosphatase has been used in conjunction with ‘‘thick section’’ electron microscopy. In resting cells, in suspension, the alkaline phosphatase is present in rod- shaped granules that can be detected by this cytochemical method only if the cells are permeabilized (Fig. 7). Fol- lowing stimulation, these cells undergo a dramatic alteration in their organization. The cells display an ela- borate system of tubular structures that are positive for alkaline phosphatase. Moreover, the cytochemical reac- tivity does not require cell permeabilization showing that the tubular structures are in continuity with the extra- cellular medium (Fig. 7). The use of ‘‘thick sections’’ in electron microcopy enables the appreciation of how dra- matic the alterations are when neutrophils are stimulated with cytochalasin B and f-Met-Leu-Phe. The full extent of these changes were not observed either with conventional thin section electron microscopy or with light microscopy (not shown). These results indicate dramatic modiﬁcations in cell shape and organization that neutrophils are capable of performing.