Power and fluorescence sensitivity of normal FCM together with the spatial resolution and quantitative morphology of digital microscopy, because it is depending on the capture of images of particles in flow and subsequent pixel-based image evaluation of objects [413]. Imaging FCM allows defining the intracellular localization of fluorescent targets in phagocytes, VEGF-A Proteins supplier therefore ruling out the need of quenching or blocking steps (Fig. 43) [41416].eight.3.6 Assessing or quantifying phagocytosis kinetics and capacity: The simplest calculation could be the proportion of phagocytosing cells within the evaluated population, defined because the percentage of gated cells with target fluorescence, present inside the proper gate(s), established by morphological, viability, and immunophenotypic criteria [377], as observed in Fig. 44. Regarding the quantification of ingested fluorescent targets, calculation might be reasonably simple if pH-independent fluorescent particles (biological or synthetic) are applied. The mean quantity of particles ingested per effector cell could be calculated by dividing the MFI with the cell population by the fluorescence of a single, extracellular target [417]. When making use of targets labeled with pH-dependent dyes, even so, this calculation is inaccurate and have to be modified by subtracting the number of no cost targets per phagocyte from the initial number of targets per phagocyte [377, 378].Eur J Immunol. Author manuscript; obtainable in PMC 2020 July 10.Cossarizza et al.FGF-2/bFGF Proteins Formulation PageAn fascinating parameter to quantify phagocytosis capacity is the phagocytosis product (PP) parameter [377]. PP is defined as the percentage of phagocytosing cells multiplied by the amount of targets per phagocytosing cell. PP reflects that the total elimination of targets from a given assay preparation depends each from the percentage of phagocytosing cells and also the number of targets ingested by each effector cell [377]. eight.4 A basic protocol for assesing phagocytosis in whole-blood samples employing pHrodo Red E.coli BioParticles 8.4.1 Overview: This assay is appropriate to decide phagocytic activity in whole blood samples depending on the usage of pHrodo E. coli Red BioParticles, which undergo a powerful boost in fluorescence when the surrounding pH becomes much more acidic through the ingestion phase of phagocytosis method. Labeling of complete blood samples with appropriate panleukocytic markers, including CD45 or CD11a (Fig. 44), easily enables excluding easily erythrocytes and platelets. Utilizing species-specific phagocyte markers permits to evaluate phagocytosis of pHrodo BioParticles by granulocytes in several species [418]. By adding a suitable fluorogenic substrate of ROS like Dihydrorhodamine 123 (DHR123), this protocol permits the simultaneous examination of phagocytosis and oxidative burst. CD11a clone HI111 reacts with human, rhesus, cynomolgus, or baboon monkey, dog, and rabbit. In addition, it has been shown in our laboratory to crossreact with some cetaceans and pinnipedes. Thus, along with human research, this protocol has been successfully applied to evaluate ingestion of E. coli and respiratory burst in whole-blood samples of dolphins (Fig. 44), Beluga whales, and walruses. eight.4.2 1. Step-by-step sample preparation and assay protocol Prepare 3 tubes and label appropriately for: two. three. 4. 5. autofluorescence control cytochalasin A (adverse control) pHrodo Red E.coli BioParticlesAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptDispense 50 L heparinized entire blood into every single tube.