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3H-thymidine content determined by liquid scintillation (2).
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14
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2642657092
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note
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null tg B cells. Mice were killed 1, 3, and 5 days after transfer. One-third of the spleen was used for two-color flow cytometry and the remainder for immunohistochemistry.
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16
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2642653045
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note
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3, 0.1 mM EDTA) to remove the remaining erythrocytes. To detect lysozyme-specific lg tg B cells, PBMCs and splenic MNCs were stained with HEL-biotin, followed by streptavidin-phycoerythrin (PE) and counterstained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) to CD45R (B220; PharMingen, San Diego, CA) [HEL-biotin was prepared with biotinamidocaproate N-hydroxysuccinimide ester (Sigma) by standard protocols]. To detect activated lysozyme-specific lg tg B cells, splenic MNCs were stained with HEL-biotin, followed by streptavidin-FITC and counterstained with PE-labeled anti-CD86 (B7-2) mAbs (all from PharMingen). The HEL-biotin probe stained only the adoptively transferred tg B cells, because no HEL staining was detected when B cells from DEL-or TEL-immune mice (where no adoptive transfer was performed) were stained with HEL-biotin. To confirm that the majority of the transferred tg B cells were detected with the HEL-biotin label over the 5-day period, a second labeling method was used. Splenic B cells from lysozyme-specific lg tg mice were labeled with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF AM) (Molecular Probes, Eugene, OR) as described (27) and transferred according to the adoptive transfer protocol. PBMCs and splenic MNCs of immune recipients were harvested as described above, stained with either HEL-biotin, followed by streptavidin-PE, or with biotinylated anti-CD45R (B220), followed bysteptavidin-PE, and analyzed by flow cytometry. Results from three to four experiments with pooled cells from two mice demonstrated similar numbers of cells positive for both HEL binding and BCECF or B220 and BCEF.
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17
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2642699665
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note
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Spleens were prepared for cryosections as described (2). For immunohistochemical staining of lysozyme-specific lg tg B cells, a two-layer sandwich of sequential incubation with biotinylated HEL was used followed by streptavidin-alkaline phosphatase (AP), and GC B cells were detected with peanut agglutinin (PNA)-horseradish peroxidase (HRP) (EY Laboratories, San Mateo, CA). Bound HRP and AP activities were visualized by reacting with 3-aminoethyl carbazole and naphthol AS-MX phosphate-Fast Blue BB (Sigma), respectively.
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19
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0026587743
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2642699664
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note
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2 fragments (Boehringer) and streptavidin-AP. BrdU-positive cells were visualized with Fast Red TR/naphthol AS-MX (AP substrate: pink color).
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25
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0019231842
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2642627463
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note
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2-lgG1 hybridoma. Supported by the Austrian Science Foundation (M.B.F.). DAAD Sonderprogramm Rheumatologie, Germany (S.G.), the National Institutes of Health (M.C.C. and G.K.), and the New York Chapter of the Lupus Foundation (M.C.C.).
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