containing gentamycin (50 mg ml21). Then, the medium was changed again to fresh media without antibiotic and the cultures were maintained for up to 8 days with medium changes every second day. Initial samples for determination of colony-forming ability of S. aureus present in culture medium and internalized by macrophages, as well as samples for microscopic examinations, PCR analysis, and hMDMs viability assessment were all taken immediately after cells were washed out of free non-phagocytosed bacteria, before medium was supplemented with gentamycin. Consecutive samples were taken every 24h for up to 8 days or until S. aureus escaped into the conditioned medium and cells were lysed as evaluated by light microscopical inspection. All samples were prepared in triplicates. Since the opsonization of S. aureus with heat-inactivated serum (inactive complement) prior to bacterial co-culturing with macrophages did not affect the rate of phagocytosis (data not show) we assumed that the presence of serum in the culture medium during phagocytosis yielded enough opsonins to facilitate the efficient phagocytic uptake of S. aureus by macrophages. Therefore all experiments described in this paper were performed without prior opsonization. IFN c treatment hMDMs were preincubated overnight with 100 U/ml interferon c (activated hMDMs). Then, the cells were exposed to S. aureus (Newman, ATCC 25923 or COL strain) at MOI 25. Simulta- neously, the non-stimulated cells were incubated with the same S. aureus strain and regarded as a control. After 2 hours of phagocytosis, extracellular bacteria were killed by adding the gentamycin to the culture medium and the cultures were continued as described above. Directly after phagocytosis and at consecutive days post-phagocytosis, intracellular bacteria released by cell lysis were analyzed for colony forming ability. Assay for colony-forming ability of engulfed bacteria PBS-washed hMDMs directly after 2h incubation with S. aureus and infected cell cultures at consecutive days post-phagocytosis were lysed by treatment with ice-cold water. The lysates were plated at serial dilutions in sterile PBS on TSB agar plates. The plates were incubated at 37uC overnight or for 2 days and the number of colonies (CFU) was enumerated. In parallel, the presence of viable staphylococci in the conditioned media was also determined by the colony-forming assay. Lactate Dehydrogenase (LDH) measurement- Cytotoxicity Assays To assess the effect of S. aureus on cell necrosis, the LDH release assay was performed using the CytoTox96 nonradioactive cytotoxicity assay kit (Promega) according to the manufacturer’s instructions. Cytotoxicity was calculated with the formula: % cytotoxicity = (experimental value2low control)6100/(high con- trol–low control), where low control is assay medium plus cells and high control is assay medium (plus 2% Triton X-100) plus cells to define the maximum LDH release. The spontaneous release was always up to 10% of the maximum release. Relative amounts of LDH release was measured (absorbance at 490 nm) using an ELISA plate reader. All assays were performed in triplicate. MTT Cell Proliferation and Cytotoxicity Assay To quantify mitochondrial activity of infected hMDMs the MTT tetrazolium dye reduction assay was applied. Cultures of control Survival in Macrophages and S. aureus infected hMDMs at different times post-phagocytosis were incubated with the MTT (monotetrazolium) reagent (Sigma) for 3 h at 37uC. Then, a detergent solution was added to lyse the cells and solubilize crystals of formazan produced by mitochon- dria. The samples were read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced was directly proportional to the number of viable cells. All assays were performed in triplicate. Respiratory burst assay Control hMDMs, and S. aureus-infected cells on 4th day post- phagocytosis, were washed twice with PBS and scraped into TBS buffer. Cell suspensions (0.56106 in 0.6 ml of TBS) were incubated at 37uC for 5 min with 10 mM 29,79-dichlorodihydro- fluorescein diacetate (DCFH-DA) obtained from Molecular Probes, Inc. (Leiden, The Netherlands). Next, live S. aureus (107 CFU) or latex beads (1.7–2.2 mm, Sphero Nile Red BD Bisciences Pharmingen) were added to macrophage suspensions. At differing time intervals aliquots were withdrawn and subjected to flow cytometric analysis. The fluorescence and light-scattering proper- ties of cells was determined using FACScan Cytometer (Becton Dickinson). The green fluorescence of DCF was observed using wavelengths of 521 nm (emission), and 488 nm (excitation). The mean fluorescence intensity of cells was quantitatively analyzed. Fluorescence staining and confocal microscopy hMDMs cultured on glass coverslips were placed into 6 well plates. Both, control and infected with S. aureus (Newman strain) cells were fixed for 30 min at room temperature with 8% paraformaldehyde in 200 mM HEPES, pH 7.4, then washed with CMF-PBS. The fixed cells were treated with RNase A at 4uC, overnight. After incubation with RNase, 10 mg ml21 acridine orange (AO) was added to the samples. Green fluorescence corresponds to nucleic acids of host cell DNA as well as to nucleic acids of bacteria. Cells were examined using the Bio-Rad MRC1024 confocal system, interfaced with a Nikon Diaphot (Nikon, Amsterdam) inverted microscope equipped with argon and krypton-argon lasers, 3 detection channels and a Nikon PlanApo 60x oil immersion lens. Images were taken in the line-averaging mode and at a pinhole setting of one Airy unit. Every fluorescence image shown is an extended focus calculated as the zero-projection from stacks of single images taken at an interval of 0.2 mm. Image resolution was 5126512 pixels. AO interacts with DNA and RNA, having green fluorescence with an emission maximum at 525 nm when bound to DNA; upon association with RNA, its emission is shifted to ,650 nm (red luminescence). Also, the cells were stained with propidium iodide (PI) at a final concentration 5 mg ml21. Propidium binds to DNA and RNA, having red fluorescence with an emission maximum at 620 nm. The cells were also stained with BODIPY493/503 at final concentration 5 mM. The BODIPY fluorophore is specific for neutral, cytoplasmic lipids and used as tracer for lipid droplets and nonpolar liquids. Images were recorded using a LSM 510 Meta confocal laser scanning microscope (Carl Zeiss GmbH, Oberkochen, Germany) equipped with helium-neon and argon lasers. To distinguish vivid bacteria with intact cell walls from dead bacteria with compromised membranes, the LIVE/DEAD BacLight Bacterial Viability kit was used. The kit provides two different nucleic acid stains-SYTO 9 and propidium iodide, which, mixed in proper ratios, give fluorescence signals indicative of alive or dead bacteria. Cells were permeabilized with 0.2% Triton X-100 to allow PI to bind to the dead bacteria and macrophage DNA (Fig. 3D). Green excitation (514 nm) light was used for SYTO 9 and PI staining. PLoS ONE | www.plosone.org 13 January 2008 | Issue 1 | e1409