Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells

Science

Volumn 274, Issue 5295, 1996, Pages 2092-2094

  Hikida, Masaki ^a   Mori, Masaharu ^b   Takai, Toshiyuki ^a   Tomochika, Ken Ichi ^a   Hamatani, Kiyohiro ^c   Ohmori, Hitoshi ^a  

^a OKAYAMA UNIVERSITY   (Japan)

^b OKAYAMA PREFECTURAL UNIVERSITY   (Japan)

^c RADIATION EFFECTS RESEARCH FOUNDATION   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

INTERLEUKIN 4;

ANIMAL CELL; ARTICLE; B LYMPHOCYTE; CELL MATURATION; GENE EXPRESSION; GENE LOCATION; GENE REARRANGEMENT; GENETIC RECOMBINATION; GERMINAL CENTER; IMMUNOGLOBULIN GENE; LYMPHOCYTE SUBPOPULATION; MOUSE; NONHUMAN; PRIORITY JOURNAL; T LYMPHOCYTE;

ANIMALS; B-LYMPHOCYTES; CELL NUCLEUS; CELLS, CULTURED; DNA-BINDING PROTEINS; GENE EXPRESSION; GENES, RAG-1; GERMINAL CENTER; HOMEODOMAIN PROTEINS; IMMUNOGLOBULIN CLASS SWITCHING; INTERLEUKIN-4; INTERLEUKINS; LIPOPOLYSACCHARIDES; LYMPH NODES; LYMPHOCYTE ACTIVATION; MICE; MICE, INBRED C3H; MICROSCOPY, FLUORESCENCE; PROTEIN BIOSYNTHESIS; PROTEINS;

ANIMALIA;

EID: 0030475466     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2092     Document Type: Article

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31. Culture plates of 100 mm diameter were coated with 100 μg/ml of mAb to mouse IgD (Biosys). Sixty million mouse spleen cells that had been depleted of T cells and erythrocytes were placed in the plate and incubated for 1 hour at room temperature. Then the plate was gently washed with phosphate-buffered saline four times to remove nonadherent cells. It was confirmed by flow cytometric analysis (FACScan) that adherent cells recovered from the plate were more than 99% positive for slgD and B220.
32. Monoclonal antibodies to mouse RAG-1 (G109-256.2, mouse IgG2b) and RAG-2 (G110-461, mouse IgG2b) were obtained from Pharmingen. A myeloma-derived murine IgG2b (MOPC 195) was used as an irrelevant negative control (ICN Biomedicals). These mAbs were biotinylated under the same conditions, using a biotinylation kit (American Qualex). Staining of RAG proteins in thymocytes or cultured B cells was carried out as described (10), with some modifications. Briefly, the cultured cells were fixed on glass slides in methanol-acetone (1:1) for 5 min, rehydrated in phosphate-buffered saline, and preblocked for 1 hour with TBST [10 mM tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] containing 1% bovine serum albumin (BSA) and MOPC 195 (50 μg/ml). The slides were then incubated in TBST containing 1% BSA and one of each biotinylated mAb (5 μg/ml) for 1 hour at room temperature. Slides were then washed three times in TBST and reacted for 1 hour with rhodaminated avidin (2 μg/ml; Sigma) in TBST containing 1% BSA. For the immunofluorescent staining of LN sections, 6-μm-thick cryosections mounted on slides were allowed to air dry for 15 min and were fixed in ice-cold acetone for 10 min. After rehydration and pre-blocking as described above, the sections were treated with biotinylated anti-RAG-1 (5 μg/ml) for 1 hour, followed by double-staining with rhodaminated avidin (2 μg/ml) and FITC-PNA (4 μg/ml; Seikagaku Kogyo) for 40 min. All reagents were diluted in TBST containing 1% BSA. After washing with TBST, samples were finally mounted with low-fluorescent glycerol and cover slip protection, and were observed with a Zeis fluorescence microscope.
33. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan. We thank H. Kagawa for excellent secretarial assistance.