*for 5?min and resuspended in 10?ml HBSS?/? followed by a second centrifugation

*for 5?min and resuspended in 10?ml HBSS?/? followed by a second centrifugation. method, we Daunorubicin measure target cell densities and antibody concentrations needed for ideal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection, and examine the connection between target cell engulfment and phagolysosomal control. We demonstrate how this approach can be used to measure unique forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantifying phagocytosis that provides opportunities to better understand the cellular and molecular rules of this fundamental biological process. coordinates over specified time intervals using the perfect focus feature of this microscope. The producing image data is definitely too vast to by hand enumerate phagocytic events across hundreds of cells and time points easily. For example, a typical time-lapse Daunorubicin video from a single field look at consists of 50C100 macrophages with as many as 20 internalization events per phagocyte. Furthermore, if we conduct a 4?h Rabbit Polyclonal to Collagen I alpha2 (Cleaved-Gly1102) experiment with an image taken once every 2 min for 12 different positions (or wells), that would involve counting 1.4106 individual engulfment events. Additionally, the use of automation reduces observer bias in the enumeration process. Therefore, we developed a user-guided, semi-automated process utilizing various functions within the Nikon NIS Elements imaging software to conduct strong enumeration of large numbers of phagocytic events in numerous macrophages across multiple samples for many hours. Finally, from these data, we developed and applied several index calculations to quantify phagocytosis. Development of high-resolution temporal measurement of discrete phagocytic events reveals a delay in dye uptake The difficulty in identifying and quantifying discrete phagocytic events in co-culture experiments by microscopy is definitely illustrated in Fig.?1C and Movie?1. As is definitely apparent in the merged image (phase and fluorescence overlaid) of the 60?min time point (Fig.?1C), non-phagocytosed target cells can cluster around individual macrophages, obscuring the macrophages and thus making recognition and counting of discrete phagocytic events very difficult. Furthermore, one can appreciate, with this field of look at, the laborious effort needed to enumerate phagocytic events for each individual macrophage across all frames over time. Our method overcomes these problems by first identifying and counting the macrophages in each image by developing a macrophage binary face mask derived from the fluorescence transmission from each macrophage in the field (Fig.?1D; Movie?2). The macrophage count allows us to normalize the measured engulfment events per field, and, consequently, compare levels of phagocytosis across a range of experimental conditions. Next, individual phagocytic events are counted mainly because voids created within dye-labeled macrophages, which is best illustrated by close-up images (Fig.?2; Movies?3C7). Actually in close-up merged images (Fig.?2A, Movie?3), the difficulty in identifying internalized (versus bound) focuses on is apparent. To resolve internalized targets, we then independent the merged images into the component channels, as demonstrated in Fig.?2A: differential phase contrast Daunorubicin (Phase, Movie?4), Daunorubicin macrophage dye label (TAMRA, Movie?5) and target cell dye label (CypHer5, Movie?6). In the Phase channel, non-phagocytosed cells obscure macrophages, making enumeration of phagocytic events extremely hard. In the TAMRA channel, only the macrophages are prominently visible and the non-engulfed target cells no longer obstruct a definite look at of the macrophage. In this situation, individual phagocytic events can be clearly visualized as dark voids within the labeled macrophages (Fig.?2A). As seen in Fig.?2A, CypHer5 transmission from the prospective cells is often problematic for ascertaining individual phagocytic events due to diffuse transmission within the macrophage, uneven labeling of target cells and transmission from unengulfed target cells overlaying macrophages, as well as the delay in kinetics relative to actual phagocytic events due to the time required for pH compartment acidification (Kapellos et al., 2016; Neaga et al., 2013). Therefore, void counts are a more sensitive and instantaneous measure of phagocytic uptake. Furthermore, imaging software can automatically count voids after generating a user-defined void binary face mask (Fig.?2B; Movie?7), which provides high-resolution temporal data (Fig.?2C, remaining). The void count kinetics.