bicarbonate-0.02 mg/ml Alexa Fluor 488 dye and incubated at room temperature for 30 min. Unconjugated dye was removed by centrifugation and the labeled spores were washed five times with sterile distilled water. Alexa Fluor 488-labeled spores were biotin labeled by resuspending the spore suspension in 0.1 M sodium bicarbonate buffer (pH 8.2)20.3 mg/ml of Sulfo-NHS-LC-Biotin (Molecular Probes, Invitrogen, U.S.A.) and incubated for 45 min at room temperature. Biotin- Alexa Fluor 488-labeled C. difficile spores were washed, counted with a Heber Bacteria Counting ChamberZ30 (Hawksley, UK) and stored at 220uC until use. The fluorescent labeling did not decrease spore viability (data not shown). Visualization of Labeled Spores by Fluorescence Microscopy To quantify adherence and phagocytosis of C. difficile spores by Raw 264.7 cells, macrophages were seeded at a concentration of 66105 cells/well onto 8 well culture slides (FD Falcon) and incubated at 37uC for 24 h. Raw 264.7 cells were washed twice with DPBS (Gibco), and incubated with 100 ml of DMEM containing biotin- and Alexa Fluor 488-labeled C. difficile spores at an MOI of 4 for 30 min at 37uC. Wells were rinsed twice with DPBS to remove any unbound spores, and fixed for 15 min at room temperature with 200 ml of freshly prepared 4% para- formaldehyde. Fixed macrophages were rinsed twice with DPBS and extracellular C. difficile spores were labeled at room temperature for 50 min with streptavidin-Alexa350 (Molecular Probes, Invitrogen, CA) diluted 1:100 in DPBS-1% Bovine Serum Albumin (Sigma-Aldrich), and rinsed three times with DPBS. Cells were permeabilized with 0.06% Triton X-100 in DPBS for 15 min at room temperature, rinsed three times with DPBS, stained for F- actin with 1 U of Alexa Fluor 568-phalloidin (Molecular Probes) for 30 min and rinsed three times with DPBS. Samples were air dried, sealed with nail polish and analyzed in a DM4008B fluorescence microscope (Leica, Wetzier, Germany). Internalized spores were identified as green spores that were not labeled blue by streptavidin-Alexa350, while extracellular or adherent spores were identified as green spores that superimpose with the macrophage’s F-actin cytoskeleton and that were stained blue by streptavidin- Alexa350. For each test conditions, pictures were taken for at least ,3000 spores in 100 fields, and photomicrographs were prepared with Adobe Photoshop and Microsoft Picture Manager Software and extracellular and internalized spores counted in by eye. All experiments were performed at least three times. To evaluate if C. difficile spores were internalized through an actin polymerization dependent mechanism by Raw 264.7 cells, 10 mM of cytochalasin D was added to each well prior to the infection, and maintained for the duration of the experiment. Infected macrophages were washed and treated for fluorescence microscopy analysis as described above. To evaluate the effect of complement on binding and phagocytosis, C. difficile spores were incubated with DMEM-10% untreated or heat inactivated fetal bovine serum (FBS) (Gibco, U.S.A.) or with DMEM-10% heat inactivated FBS supplemented with 3–4 week rabbit complement (diluted 1:50 in heat inactivated FBS) (Biotech Brand) for 30 min prior to infection. Infected cells were washed and treated for fluorescence microscopy as described above. Killing of C. difficile Spores by Raw 264.7 Cells To quantify killing of C. difficile spores, macrophages were seeded 24 h prior to infection in 24-well plates. Macrophages were rinsed with DPBS and infected with 200 ml of DMEM containing C. difficile spores at various MOI’s. After 30 min of incubation at 37uC, infected macrophages were washed three C. difficile Spores and Macrophage Interactions times with DMEM, resuspended in 400 ml of DMEM in absence of FBS and incubated for various periods of time under aerobic conditions at 37uC in 5% CO2. Viability of C. difficile spores was determined at 0.5, 5, and 24 h after infection by lysing infected macrophages with 0.01% Triton X-100, serially diluting into DPBS and plated onto BHI agar plates (Difco) supplemented with 0.1% sodium taurocholate (ST) and in- cubated for 24 hrs anaerobically at 37uC for colony counts, no additional colonies were observed upon further incubation periods. In some experiments, C. difficile spores were pre- incubated with or without 1.0% ST, 0.1% ST-5 mM L-glycine (STG), 50% human serum (HS) in DMEM, or with 50% HS- STG for 30 min before infecting Raw 264.7 cells. Initial spore counts were quantified by plating serially diluted aliquots onto BHI agar plates. To evaluate outgrowth of STG-treated C. diffcile spores during infection of macrophages, Raw 264.7 cells were infected for 30 min with STG-treated spores incubated for 24 h under either aerobic with 5%-CO2 or anaerobic conditions in a anaerobic chamber Bactron III-2 (OR, U.S.A.), fixed with paraformaldehyde as described above, stained with DAPI and examined by fluorescence microscopy. Killing of C. difficile Vegetative Cells by Raw 264.7 Cells To quantify killing of C. difficile vegetative cells by Raw 264.7 cells, C. difficile cells were quantified in overnight cultures (,18 h) with a Heber Bacteria Counting Chamber Z300 (Hawksley, UK) and used to infect at an MOI of 10 as described above. Infection under aerobic conditions was done with 5%-CO2, while for anaerobic conditions, Raw 264.7 cells were preincubated for 1 h in an anaerobic chamber (Bactron III-2, Shellab, OR, U.S.A.) in DMEM medium that had been previously reduced for 72 h under anaerobic conditions, and infected with C. difficile cells resuspended in prereduced DMEM medium. All manipulations requiring anaerobic conditions were done inside a Bactron III-2 anaerobic workstation (Shellab, OR, U.S.A). Initial cell counts were quantified by plating serially diluted aliquots onto BHI agar plates. Live/dead Assay of Raw 264.7 Cells To evaluate the cytotoxic effects of C. difficile spores on Raw 264.7 cells during the course of infection, Raw 264.7 cells (,56105) seeded in 96 well plates were infected with C. difficile spores at various MOI for 30 min. Raw 264.7 cells were washed twice with DPBS to remove unbound cells and infected monolayers of Raw 264.7 cells were incubated in DMEM in absence of FBS under aerobic conditions with 5% CO2 for 24 and 48 hrs. Cytotoxicity was evaluated using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, OR, U.S.A.). Live cells are distinguished by the enzymatic production of intensively fluorescent calcein (Ex/Em 494/517 nm) from non-fluorescent cell-permeant calcein AM through intracellular esterease activity. Dead cells are detected with ethidium homodimer-1 (Ex/Em 528/617 nm) that enters cells with damaged membranes and binds to nucleic acids increasing 40-fold its fluorescence intensity, producing a bright red fluorescence in dead cells. In live cells, ethidium homo- dimer-1 is excluded by the intact plasma membrane. Inherently low levels of background fluorescence were detected in control treatments (data not shown). Dead Raw 264.7 cell control were Raw 264.7 cells treated with 0.06% Triton 100610 min prior to addition of ethidium homodimer-1, while live Raw 264.7 cell control were monolayers of Raw 264.7 cells incubated for 24 and 48 h in absence of C. difficile spores. Fluorescence was measured in a Infinite 200 PRO (Tecan, U.S.A.) using PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43635