Multiparameter flow cytometric kinetics of phagocyte stimulus responses.

THE recently published Technical Note by Vines et al., ‘‘A Flow-Cytometric Method for Continuous Measurement of Intracellular Ca Concentration,’’ Cytometry Part A 77A:1091 1097, 2010, demonstrated that such measurements are possible in an Accuri C6 flow cytometer. Their contribution demonstrates that rapid measurements can be achieved when stimuli are added to cells (in an open tube) which then flow via a pump mechanism, yielding observations within 5 s, judging from their figures, albeit without thermostating or stirring the sample. Intracellular kinetic measurements of cytoplasmic Ca (and other events) using other flow cytometers (FACS 440, MoFlo, Aria) have been previously reported by a number of laboratories, including ours and extensively reviewed (1 9). Since our own first report in 1986 (10), we have published numerous flow cytometric measurements of cytoplasmic Ca fluxes ([Ca]in) in various blood cells, using a modified FACS 440 (BD) or MoFlo (Cytomation), injecting the compounds into a stirred thermostated flowing cell suspension. These flow cytometers, and the LSRII and FACSAria to which we are now adapting the sample handling system we described, use N2 pressure to move the cell stream, which permits the sample to be stoppered and reduces risk from potential biohazards. Thermostating and stirring assure physiologic conditions and rapid mixing. Injecting into pressure-driven, flowing cells a very short (<10 cm) distance from the nozzle, permits continuous observation of cellular responses within 4 s after reagent addition without using high flow rates. Using the technique above, we also have correlated cytoplasmic changes in Ca and pH, and consequent reactive oxygen and lytic entities, with specific receptor occupancy and subpopulation responses by and with the neutrophils’ receptor cross-linking requirements (11). Similar studies performed with monocytes (12) permitted differentiation between cells responding and not responding withD[Ca] by the presence or absence of a specific receptor (CD14) on their surface (Fig. 1). Our flow cytometric methods have now been applied to studies of the early kinetics of cytoplasmic and phagosomal changes as cells respond to stimulating organisms (e.g., changes in phagosomal pH, generation of reactive oxygen species (ROS), presence of lytic enzymes) by labeling the phagocytized entity so that it acts as an indicator of conditions in that compartment (11). Recently, we showed that neutrophil (PMN) stimulation by Staphylococcus epidermidis (13) requires opsonization of the organism. A transient spike in D[Ca]in is maximal within 18 s and originates from intra PMN stores. Using pHrodo (LifeSciences), we further showed that the phagosome enclosing S. epidermidis becomes increasingly alkaline, in contrast to the acidification we observed when a fungus, Cryptococcus neoformans, was phagocytized. By using flow cytometry and our adaptation for rapid kinetics (described above) in conjunction with multiple labels on the same organism, we were able to correlate the initial D[Ca]in signal not only with the phagosomal pH (pHp) but also with the ROS, generated in the phagosome and the receptor occupancy on the same PMN. Quenching the fluorescence of bound but not yet phagosome-enclosed S. epidermidis permitted us to focus on the conditions within it and to begin investigation of the role of extracellular Ca. This method gains greater potential relevance when it is used to investigate the very early, coordinated signaling events that occur upon binding and phagocytosis of organisms like mycobacteria that are able to alter the phagocyte response to evade killing and establish latent infection. Binding-induced fluxes resulting in rapid changes in local ion concentrations and their relationships to activation of membrane-localized