incubator at 37uC as described previously [32,33]. Six days after initial BM cells culture, the medium was changed and the purity of F4/80+ cells was more than 90%, as determined by flow cytometry (FACSCalibur; BD Biosciences). DNA preparation ALD-DNA and UnALD-DNA were prepared with murine splenocytes which were generated from surgical resected spleens of six- to eight-week-old female BALB/c mice and cultured with or without Con A (Sigma-Aldrich) in vitro as previously described [7]. Briefly, for generation of ALD-DNA, splenocytes were seeded at 26106 cells/ml in 75 cm2 cell culture flask and cultured in the presence of Con A (5 mg/ml) for 6 days to induce apoptosis. The apoptotic cells were stained with FITC-labeled Annexin V (BD Biosciences) and propidium iodide (PI; Sigma-Aldrich), and sorted using a FACSAria (BD Biosciences). Genomic DNAs from syngeneic apoptotic splenocytes were treated with S1 nuclease (TaKaRa) and proteinase K (Sigma-Aldrich), and then purified using the DNeasy Blood & Tissue Kits (Qiagen) according to the manufacturer’s instructions. UnALD-DNA was prepared with unactivated (resting) splenocytes and extracted using the same methods. To exclude contaminations with LPS, sterile endotoxin-free plastic ware and reagents were used for DNA preparation. DNA samples were also monitored for low level of endotoxin by the Limulus amoebocyte lysate assay (BioWhittaker) according to the manufacturer’s instruc- tions. The concentration of DNA was determined by detection of the absorbance (A) at 260 nm. The apoptotic DNA ladder of ALD-DNA was confirmed by agarose gel electrophoresis (AGE). Generation of SLE murine model To generate SLE murine model, 6- to 8-wk-old syngeneic female BALB/c mice were divided into several groups of 8–10 mice and actively immunized by subcutaneous injection on the back with 0.2 ml of an emulsion containing ALD-DNA (50 mg/ mouse) in phosphate-buffered saline (PBS) plus equal volume of complete Freund’s adjuvant (CFA; Sigma-Aldrich) at week 0, and followed by two booster immunizations of ALD-DNA (50 mg/ mouse) emulsified with IFA (Sigma-Aldrich) at week 2 and week 4 for total 3 times as previously described [6,7]. Eight to 10 mice in each group received an equal volume of PBS plus CFA or IFA, or UnALD-DNA (50 mg/mouse) plus CFA or IFA were used as contols. Mice were bled from retro-orbital sinus prior to immunization and at 2-week internals until 3 months after the initial immunization. 8 or 12 weeks later, mice were sacrificed and surgical resected spleens and kidneys were collected for further cellular function and tissue histology analysis. Autoantibody and proteinuria examination Anti-dsDNA antibodies in the mice serum were determined by ELISA assay as described previously [6]. In briefly, ELISA plates (Costar) were pretreated with protamine sulphate (Sigma-Aldrich) and then coated with calf thymus dsDNA (Sigma-Aldrich). After incubation with mouse serum, the levels of anti-dsDNA Abs were detected with the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech). Tetramethylbenzidine (TMB) substrate was used to develop colors and absorbance at 450 nm was measured on a microplate reader (BIO-TEK ELX800). Proteinuria of the mice was measured with the BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Measurement of anti-dsDNA antibody titers Anti-dsDNA antibody titers in the mice serum were determined by ELISA assay as described previously [34]. In briefly, protamine Reversal of Lupus Nephritis by SAP Treatment sulphate pre-treated 96-well microtitre plates (Costar) were coated with calf thymus dsDNA (Sigma-Aldrich; 50 mg/ml) for 2 h at 37uC and then placed overnight at 4uC. After washing three times with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h, and serial dilutions of serum in PBS–1% BSA were added for 1 h at 37uC. After washing, the plates were incubated with 1:1000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech) for 1 h at 37uC. Tetramethylbenzidine (TMB) substrate was used to develop colors and absorbance at 450 nm was measured on a microplate reader (BIO-TEK ELX800). Measurement of SAP level To assess protein levels of SAP in serum of mice or in the culture supernatants, ELISA assays were performed with the following anti-SAP Abs and SAP standards: sheep anti-mouse SAP (Calbiochem), rabbit anti-mouse SAP (Calbiochem), and mouse SAP (Calbiochem) as previously described [35]. Measurement of circulating DNA level DNA was extracted from serum samples and then quantified using a PicoGreen DNA detection kit (Invitrogen) according to the manufacturer’s instructions [36]. In briefly, DNA was extracted from 200 ml of serum samples using a QIAamp Blood Kit (Qiagene) using the blood and body fluid protocol as recom- mended by the manufacturer. After the removal of most proteins by digestion with proteinase K, the sample was applied to the QIAamp 96 plate. DNA was adsorbed onto the silica membrane during a brief centrifugation step, while any remaining protein, salt and other contaminants were completely removed by three consecutive washes. Membrane-bound DNA was then eluted in double deionized H2O or Tris–EDTA buffer. A final elution volume of 200 ml was used. Quantification of DNA was carried out using a PicoGreen DNA detection kit (Invitrogen). Calf thymus DNA (100 mg/ml; Sigma-Aldrich) was used as the standard. The concentration of DNA in the standard curve ranged from 0 to 100 ng/ml. Briefly, 20 ml of final DNA eluated was mixed with 1 ml of Tris–EDTA (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 7.5) diluted with PicoGreen reagent. Fluorescence intensity was measured on an F-2000 spectrofluorometer (Molecular Devices) at excitation wavelength of 480 nm and an emission of 520 nm. Standard curve used to determine the levels of circulating DNA in the samples was established by the linear relationship between the known concentrations of calf thymus DNA (Sigma- Aldrich) and the corresponding fluorescence intensities. Immunohistochemistry examination The expression of SAP protein in the muscle tissue received pcDNA3-SAP (pSAP) injection was analyzed by immunohisto- chemistry. In briefly, mice were injected with pSAP (100 mg/mice) at the site of femoral muscle. 3 days later, the muscle tissue harvested from pSAP treated mice were fixed in 4% paraformal- dehyde, processed on a standard histology processor, embedded in paraffin, and cut into 5 micron sections. Paraffin sections were dewaxed in xylene and rehydrated in decreasing concentrations of alcohol. Sections were exposed to citrate buffer and heat antigen retrieval and then blocked and incubated with rabbit anti-mouse SAP antibody (Calbiochem). Sections were subsequently assayed with the Super Sensitive Polymer-HRP IHC Detection System (Vector Laboratories) according to the manufacturer’s instructions. 3, 39-Diaminobenzidine (DAB) substrate (Dako) was used to develop slides. Slides were counterstained with hematoxylin (Dako) and coverslipped using Permount mounting media (Fisher Scientific). Pictures were acquired with a 206/0.45 Plan Fluor PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22659