A quantitative approach to correlate astrocyte differentiation and phagocytic activity

A triple staining procedure (PAP labeling for GFAP, PAS reaction for added yeast cells and hematoxylin for nuclear staining of the whole cell monolayer) had disclosed that Junin virus infection enhanced phagocytic activity by inducing greater astrocyte differentiation. Here, we resorted to a mathematical approach for simultaneous evaluation of astrocyte differentiation and potential phagocytosis. At light microscopy level, the total number of: a) PAS-stained yeast cells, b) PAS-stained yeast cells associated to GFAP-positive astrocytes, c) GFAP-positive astrocytes, and d) total number of GFAP-labeled and non-labeled astrocytes, were counted within the monolayer area delimited by a grid with a total area of 0.01 mm2. As the percentage of PAS-stained yeast cells associated to GFAP-positive astrocytes correlated significantly with the percentage of GFAP-positive astrocytes for the three yeast cell incubation times (24, 48 and 72 h), a mathematical approach involving a so-called beta parameter representing the percentage of differentiated astrocytes capable of taking up 50% of added yeast cells, was developed. Since beta value dropped along yeast cell incubation time, and more markedly in Junin-virus infected samples, a numerical value was thus available to assess enhanced phagocytic activity in astrocytes undergoing differentiation. Therefore, the application of a mathematical approach to cell monolayers subjected to current staining techniques, allows more objective analysis of data provided by cursory visualization at light microscopy level.