x ELISA plate reader. For 
each of the cytokines, appropriate standards and blanks were used. Phagocytosis assay Fluorescent yellow green latex beads of size 2 m were added at a concentration of 10 g/ml to APECs and incubated for 30 min at 37C. Further the cells were washed with PBS to remove any excess beads that remained extracellular, before staining with bisBenzamide. The amount of total fluorescence was obtained using Infinite 200 pro fluorescence microplate reader and represented fold change with respect to the untreated C57BL/6 APECs. Statistical analysis All the data were plotted using GraphPad Prism 5 software and the data is presented as the mean standard error. The significance was obtained by performing one way analysis of variance among the test groups and p-values less than 0.05, 0.01 and 0.001 have been represented as , and respectively in comparison to untreated control. In case other controls were used for comparison, the usage of additional symbols for the same are mentioned in the figure legends. Results Ifn induces aggregation of APECs in a CD11b-dependent manner We have been interested in elucidating novel functional responses to Ifn by tumor cells and primary APECs. As observed in Fig 1A, there are very few T cells or neutrophils in APECs which predominantly consists of ~80% macrophages as indicated by the markers such as MHC class II, CD11b and F4/80. However, a minor population of B220 positive cells was 6 / 28 Ifn and Nos2 Regulate Functions of APECs Fig 1. Ifn induces APECs to aggregate. APECs were isolated from C57BL/6 mice, cultured in tissue culture media for ~ 24 h and were characterized by staining with a panel of antibodies to different cell surface proteins followed by FACS analysis. Representative plots and data for different markers is shown which are representative of multiple experiments with APECs from 4 mice. Bright field images of APECs treated with different doses of Ifn for 36 h and kinetics of APECs treated with 25 U/ml Ifn for indicated time points. The scale bar represents 20 m. Quantification of the extent of APECs aggregates formed as a function of dose of Ifn and incubation time. An aggregate consists of six or more interacting cells in any given field acquired at 20X magnification. The data is represented as mean S.E from three independent experiments. doi:10.1371/journal.pone.0128301.g001 also present in our culture system. We observed that addition of Ifn in a dose and time dependent manner increased the formation of aggregates of APECs. These are defined as clusters of six or more cells in close association with each other in a given field at a given time point. As cell-cell interactions often involve adhesion molecules, we investigated the possible regulation and roles of cell surface selectins and integrins. Ifn is known to induce the SB 203580 expression of MHC molecules and modulate the expression of adhesion molecules. Therefore, the quantitative expression of different cell surface molecules in response to Ifn PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697345 treatment was analyzed in a kinetic manner. The addition of Ifn did not affect cell surface amounts of P-Selectin, but led to an increase in cell surface expression of MHC class II 7 / 28 Ifn and Nos2 Regulate Functions PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698726 of APECs molecule. However, Ifn led to a modest decrease in the expression of Icam1, Lymphocyte function associated antigen 1, E-selectin and CD11b. Further, fluorescence microscopic analysis of APECs in response to Ifn treatment revealed preferential cell surface loca