In the present article, we have focused on developing protocols to study these early signaling events during phagocytosis of microorganisms by using the organism itself as reporter of conditions within the phagosome. Multiparameter kinetic Sunitinib order
flow cytometry permits the simultaneous real-time analysis of multiple events and of correlating these with receptor occupancy on a cell-by-cell basis. Furthermore, they allow the detection of subpopulations, responding vs. nonresponding, and thereby of distinction between dose-dependent responses by all the cells vs. maximal responses by a dose-dependent subpopulation and a lack of response by the rest of the cells (15). Our protocols use organisms, labeled with multiple fluorescent probes, as stimuli, injected into thermostated, stirred phagocyte suspensions, with data recorded continuously. By modifying the sample supply systems of our flow cytometers, the time delay from injection to appearance of phagocyte-bound fluorescent organisms can be reduced to <7 s, limited only by the length of the sample delivery tube and the flow rate the phagocytes can tolerate without shear problems. This is sufficient to capture very early events in the cell signaling process. Our current studies query the pHp, which is critical to antibacterial function of the phagocyte. In previous article using fluorescein-labeled organisms and image analysis, we showed that C. neoformans creates <a href="http://www.selleckchem.com/products/3-methyladenine.html
">3-Methyladenine molecular weight a hyperacidic phagosome in human PMN, a condition under which the normal pH-dependent neutrophil bactericidal/fungicidal entities do not work (24). We confirmed that creating an excessively low pHp was indeed the evasion mechanism by restoring Cf killing when the phagosome was alkalinized with chloroquine (24). Better tools for studying pH changes in the cell over time are now available. The novelty of those studies, carried over into the present work, lay in using the organism itself as an indicator, rather than employing inert indicators, such as fluorescently labeled beads or particles (e.g.,26, 45) or inert protein substrates (26) used by others. Furthermore, several of those studies preloaded the indicating particles into the phagocytes, i.e., ��priming�� them, and then Ceftiofur
observed the resultant changes fluorescently, but always after long time intervals. For example, using fluorescein-labeled particles and live, labeled bacteria, Sturgill-Koszycki et al. (27), have reported that phagosomes of murine MP had not acidified several minutes after ingesting nonpathogenic Mycobacteria avium. This response has been attributed to the failure of the cell's ATPase to localize to the phagosome membrane (20, 27), and may represent a unique outcome for this organism. These studies used a fluorescein derivative whose pH sensitivity, as we have shown here, drops sharply above pH 7.