as shown for SP-D [44,45]. On the other hand, the redundancy may stem from down-regulation of this receptor during mycobac- terial infection, as macrophage activation has been shown to suppress SR-B1 expression [30]. Furthermore, expression of the related human scavenger receptor CD36 has been found to be lower on monocytes in TB patients, and reverse to normal levels upon anti-mycobacterial treatment [46]. At the same time, when resident alveolar macrophages differentiate by cytokine stimula- tion, they increase the expression of surface receptors and show a higher affinity to bind mycobacteria [27], therefore maybe completely render any role of SR-B1 redundant. Materials and Methods Cell Culture NIH3T3 murine embryonic fibroblasts (ATCC), RAW264.7 murine macrophages (ATCC), rat 6 fibroblasts (R6F, [47]) and the packaging cell line plat-E [48] were grown in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Delta Bioproducts), 2mM L-glutamine, 100U/ml penicillin and 100mg/ ml streptomycin (Sigma) in a humidified atmosphere (5% CO2) at 37uC, if not otherwise indicated. Ligand Binding Assays The cells used for the binding assays were plated in 24-well- plates at a density of 56104 cells per well and allowed to adhere overnight. Nonopsonic binding studies were performed with live strains of Mycobacterium bovis bacilli Calmette-Gue´rin (BCG) expressing either Green Fluorescent Protein (BCG-GFP) or bacterial luciferase (BCG-lux) as well as Mycobacterium tuberculosis H37Rv expressing bacterial luciferase [49] (Mtb-lux) and FITC- labeled zymosan (Molecular Probes). Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10mg/ml kanamycin (BCG-GFP) or 50mg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at 280uC at a density of approximately 56106 particles. For binding studies, BCG or Mtb aliquots were thawed, spun in a microcentrifuge for 1 min at 13000g, washed twice in DMEM followed by vigorous vortexing, and aggregates were dispersed by sonication for 30 sec at 50W in a cuphorn sonicator. To infect cells, culture medium was removed, and mycobacterial suspension in serum-free DMEM (if not otherwise indicated) was added at a particle:cell ratio of 10:1. Where indicated, binding was performed in the presence of additives such as 10% serum, 1:100 diluted rabbit anti-mouse SR- B1 polyclonal antibody (NB 400-134, Novus Biologicals), 100mg/ ml LDL, 50% bronchoalveolar lavage fluid (BAL), or methyl-b- cyclodextrin (MbCD, Sigma), respectively, at the indicated concentrations. FITC-labeled zymosan was prepared similarly, sonicated for 3 min at 150W and added to the indicated cells at a particle:cell ratio of 10:1. The cells were then incubated with their ligands for 1 hr at 37uC, washed three times with DMEM and prepared for further analysis. Quantification of BCG- and Zymosan Binding Binding of BCG-lux to the cells was determined by lysis of the cells in 150ml 0.1% triton X-100 and addition of 20ml 1% n-decyl- aldehyde to initiate the luciferase reaction. Light emission was measured in white 96-well plates for 2.5 sec using a Fluoroscan Ascent FL (Thermo Fisher Scientific, Waltham, MA). Binding of FITC-zymosan was quantified by lysis of the cells in 100ml 3% triton X-100. The relative fluorescence was then determined in black 96-well plates using the Fluoroscan Ascent FL. Both BCG- and zymosan binding data were normalized to cell numbers by M. tuberculosis and SR-B1 fluorescently staining an independent set of samples with 5mM CFSE (Molecular Probes) in PBS for 5 min at room temperature. After three washes cells were lysed in 100ml 3% triton X-100 and fluorescence was determined as above. All experiments were performed in triplicates, repeated at least three times and calculated as means6S.E. Values were expressed as relative light units (R.L.U.) or relative fluorescence units (R.F.U.), respectively, or as x-fold increases compared to controls. Statistical significances were calculated using the Student’s t test (*, p,0.05). Expression and Functional Screening of a Retroviral cDNA Library A retroviral cDNA expression library generated from RAW264.7 macrophages cloned into the retroviral vector pFB-Neo (Stratagene) was used for stable transduction of NIH3T3 cells as described [24]. Briefly, 16106 plat-E cells were transfected per 1mg DNA using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer’s instruction and incubated for 24 hr at 37uC, followed by another 24 hr incubation at 32uC. Retroviral superna- tants were harvested and used to transduce 16105 NIH3T3 cells in the presence of 5mg/ml polybrene (Sigma), which were then selected with 0.4mg/ml G418 (InvivoGen). The screening for cells expressing any mycobacterial-binding receptors was done by fluorescent microscopy as described in detail in [8] on a Zeiss inverted microscope Axiovert 40 CFL after incubation of the stably transfected NIH3T3 cells with M.bovis BCG expressing GFP prepared as described above. Positive cells were ring-isolated using cloning cylinders, expanded in culture for 4–7 days and re-screened until the majority of cells were found to be positive. Viral cDNA inserts were recovered from the genomic DNA of positive cells by conventional PCR using the primers 59Retro and 39pFB-Neo (Stratagene) and re-cloned into pFB-Neo for functional testing and sequencing. DNA Constructs and Generation of Stable Cell Lines The 2.5kb cDNA of SRB1 was derived from the functional screen and subcloned into the retroviral vector pFB-Neo. Subcloning of SIGNR1 in pFB-Neo has been described before [25]. Stable NIH3T3 or R6F cells expressing SRB1 or SIGNR1 were generated by retroviral transduction as described above and grown in the presence of 0.4mg/ml G418 (InvivoGen). Western Blotting Cells were lysed in Nonidet P-40 buffer (1% Nonidet P-40, 150mM NaCl, 10mM EDTA, 10mM NaN3, 10mM Tris-HCl (pH 8), 2mM Na3VO4, 10mM NaF, and complete EDTA-free protease inhibitors (Roche Applied Sciences) and incubated on ice for 30 min. Nuclei and cell debris were pelleted at 12000g for 20 min at 4uC, and supernatants were prepared for SDS-PAGE and Western blotting according to conventional protocols. Rabbit anti-mouse SR-B1 IgG (NB400-104, Novus Biologicals) and goat anti-rabbit IgG horseradish peroxidase conjugate (Jackson Im- muno Research Laboratories, Inc.) were used to detect SR-B1 using ECL chemiluminescence substrate (Amersham Biosciences). As a loading control the blots were probed with a rabbit anti-mouse GAPDH monoclonal antibody (Cell Signaling Technology, Inc.). Flow Cytometry Flow cytometry was performed according to standard protocols at 4uC. Briefly, adherent cells were lifted in 4mg/ml lidocaine hydrochloride (Sigma) in PBS supplemented with 10mM EDTA, washed in FACS wash solution (0.5% BSA in PBS) and placed at a density of 16105 cells in 96-well V-bottom plastic plates. Cells PLoS ONE | www.plosone.org 11 December 2009 | Volume 4 | Issue 12 | e8448