Meeting Abstract

P3-121   -   Flow cytometry-based analysis of phagocytic activity in leukocyte populations of Xenopus laevis Nadjarian, AN*; Timilsina, S; Raffel, TR; Oakland University, Rochester, MI; Oakland University, Rochester, MI; Oakland University, Rochester, MI andreanadjarian@oakland.edu

Ecoimmunologists who are interested in measuring environmental effects on cellular immune functions, such as phagocytosis, often work with non-model organisms and run experiments requiring large sample sizes. Traditional methods measuring phagocytosis rates by leukocytes tend to be time-intensive (e.g., microscopy) or fail to differentiate important cell classes. Here we describe the development and validation of a flow cytometry-based procedure to analyze the phagocytosis of microscopic fluorescent beads by granulocytes and B cells in a model frog species. We collected blood from adult X. laevis, isolated leukocytes by centrifuging across a percoll gradient, incubated cells with fluorescent beads for 18 hours, and analyzed bead uptake by different cell types using flow cytometry and confocal microscopy. We differentiated cell types using a B-cell specific antibody label and forward/side scatter (flow) and morphology (microscope). Flow analysis revealed clear peaks in cellular fluorescence that corresponded to integer numbers of beads, allowing us to convert continuous fluorescence data into count distributions of beads per cell for each leukocyte population. These results compared well with similar count distributions generated using traditional microscopy. We also found that the differentiation of cell types could be improved by accounting for increased forward and side scatter in cells that engulfed beads, which corresponded to the number of beads engulfed. This flow-cytometry based method will make it easier to quickly generate experimental data measuring effects of environmental factors on cellular function of specific leukocyte populations of X. laevis or related frog species.