The cells were then counterstained with DAPI and visualized using a fluorescence microscope

of combined immunotherapy targeting both plasma cells and their precursors may 2 / 17 Long-Term Plasma Cell Depletion Ameliorates SLE provide useful information for the development of therapeutic concepts in SLE and other antibody-mediated diseases. Methods Mice Female NZB/W F1 mice were bred and maintained in specific pathogen-free conditions at the mouse facility of German Rheumatism Research Centre in Berlin, Germany. All animal procedures were approved by the local authority for animal research procedures, the State Office of Health and Welfare of Berlin, Germany. Depletion regimens The following antibodies were used for treatment: mouse anti-mouse CD20-specific mAbs, anti-VLA-4 and anti-LFA-1 blocking mAbs were purified from hybridoma and bortezomib was purchased from Millennium Pharmaceuticals. For standardization, the same doses, routes and times of administration of depletive agents were used in the respective groups; anti-CD20, bortezomib and co-injection of anti-LFA-1 and anti-VLA-4. Short-term initial B- and plasma-cell depletion. To compare the efficiency PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19724269 of different short-term B- and plasma-cell depletion regimens, 20- to 22-week-old NZB/W F1 mice were fed bromodeoxyuridine dissolved in drinking water containing 1% glucose for a period of two weeks, starting one week before treatment. Mice were divided into five groups and treated with a) vehicle, b) anti-mouse CD20 antibody, c) anti-mouse CD20 plus anti-LFA-1/anti-VLA-4 blocking antibodies, d) anti-mouse CD20 combined with bortezomib, or e) anti-mouse CD20 together with bortezomib and antiLFA-1/anti-VLA-4 antibodies. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723701 All drugs were diluted in PBS. Seven days after the start of treatment, the mice were sacrificed by cervical MedChemExpress c-Met inhibitor 2 dislocation, and their bone marrow and spleens were harvested for flow cytometric and ELISPOT analysis. Short-term B- and plasma cell-depletion followed by continuous BCD therapy. Sixteen-week-old mice received either a) no treatment, b) anti-mouse CD20 plus bortezomib, or c) BCD therapy with anti-mouse CD20 or, d) treatment b followed by continuous BCD therapy with antimouse CD20. Flow cytometric analysis Fluorescence-activated cell sorting staining was performed as described previously. For flow cytometric analysis of plasma cells, after surface staining with anti-CD138, we performed intracellular staining for immunoglobulin -light chain and intranuclear BrdU staining using the BrdU-Flow-Kit according to the manufacturer’s instructions. To analyze plasma cell subsets, we stained intracellular polyclonal IgG and IgM. The antibodies used for B cell staining were anti-CD24 and anti-CD117 mAbs from BD Pharmingen, anti-IgM, anti-CD19, anti-CD93 and anti-CD21 from Biolegend, and anti-IgD, anti-CD23, anti-B220 and GL-7 3 / 17 Long-Term Plasma Cell Depletion Ameliorates SLE antibodies from DRFZ. The antibodies for the analysis of T cell subsets included anti-CD4, anti-CD8, anti-CD62L and anti-CD69 all from e-Bioscience, anti-CD3, anti-Ly6C, and anti-CD44. Cells were acquired using a FACS BD Canto flow cytometer and analyzed using FlowJo software. Absolute cell numbers were calculated based on population frequencies and total cell numbers per organ. The percent remaining cells was then determined by dividing the absolute number of cells in each treated mouse by the mean count obtained in the PBS-treated group and multiplying by 100. Detection of antibody-secreting cells by enzyme-linked immunospot assay For detection of anti-double-stranded DNA antibody-secre