Survival in Macrophages Figure 1. Internalization of S. aureus strain Newman does not affect hMDM viability until the plasma membrane is permeabilized. A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the Materials and Methods section. All scale bars = 10 mm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and 67,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] 6 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate. doi:10.1371/journal.pone.0001409.g001 PLoS ONE | www.plosone.org 3 January 2008 | Issue 1 | e1409