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 (