A role for CD81 in early T cell development

Science

Volumn 271, Issue 5246, 1996, Pages 198-200

  Boismenu, Richard ^a   Rhein, Maria ^a   Fischer, Wolfgang H ^b   Havran, Wendy L ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CD4 ANTIGEN; CD8 ANTIGEN; MONOCLONAL ANTIBODY; T LYMPHOCYTE ANTIGEN; T LYMPHOCYTE RECEPTOR DELTA CHAIN; T LYMPHOCYTE RECEPTOR GAMMA CHAIN;

ARTICLE; CELL INTERACTION; CELL MATURATION; FETUS; FIBROBLAST CULTURE; LYMPHOCYTE DIFFERENTIATION; ORGAN CULTURE; PRIORITY JOURNAL; STROMA CELL; T LYMPHOCYTE; THYMOCYTE; THYMUS;

EID: 0030022628     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5246.198     Document Type: Article

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27. The generation of mAb 2F7 will be described in detail elsewhere (6). Briefly, an Armenian hamster was immunized with the PAM epithelial cell line and spleen cells were fused with SP2/0 myeloma cells Hybridomas secreting mAb reactive with PAM cells were isolated and cloned for further study. FTOC was performed as described (27) Briefly, fetal thymus lobes were removed from embryos at day 14.5 of gestation and cultured on polycarbonate filters supported on surgical Gelfoam (Upjohn, Kalamazoo, MI) in 5 ml of complete Dulbecco's minimum essential medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C After 7 days of culture in medium alone or with mAb (25 μg/ml), the lobes were disrupted and the live cells were counted and then stained with the appropriate conjugated antibodies followed by FACS analysis.
28. 10), resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). The electroblotted 25-kD protein was excised after visualization with Amido black 10B (Bio-Rad). In situ digestion with trypsin was performed as described previously (23). Briefly the excised band was treated with polyvinylpyrrolidone to prevent binding of the enzyme to the membrane Digestion with trypsin {1 μg of 30 μl of 0.1 M TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 8.0)]} was allowed to proceed overnight The supernatant was then fractionated by reversed-phase high-performance liquid chromatography Fractions were collected manually based on absorbance at 210 nm A fraction corresponding to a symmetrical peak was subjected to chemical sequence analysis on an ABI 470A protein sequencer (Applied Biosystems, Foster City, CA), and a unique sequence was obtained. The sequence was compared to other known protein sequences with the BLAST program (24)
29. Paratormaldehyde-fixed and polyester wax-embedded (25) day 14.5 whole embryo sections (10 μm) were hydrated in ethanol, soaked in 0.1 M citrate buffer (pH 6 0), and subjected to microwave treatment for 2 min on the highest setting (Radarange 1000W; Amana, IA) This treatment substantially improved the staining intensity obtained with mAb 2F7 The endogenous peroxidase activity was blocked, and the sections were incubated for 30 min in 2% FBS. The sections were then stained for 2 hours with biotinylated mAb 2F7, adjusted to 10 μg/ml in 1% FBS, and washed Binding was visualized with streptavidin-labeled peroxidase (Jackson ImmunoResearch), followed by incubation in metal-enhanced diaminobenzidine (Pierce). No staining was observed when mAb 2F7 was omitted from the procedure. The sections were counterstained with hematoxylin, dehydrated in ethanol, cleared with Hemo-De (Fisher Scientific, Pittsburgh, PA), and mounted with DPX reagent (British drug house).
30. + cells, as evaluated by FACS analyses Cells were deposited in 1-μl drops on the surface of polycarbonate filters supported with surgical Gelfoam (Upjohn) in complete DMEM-10% FBS (5 ml) and cultured for 5 days. After culture, cell pellets were disaggregated by resuspension in complete DMEM-10% FBS and stained with phycoerythrin antibody to CD4 (anti-CD4) and Red 613 antibody to CD8 before FACS analysis.
31. We thank S Aaronson and L. Feng for providing cell lines; A. M. Gonzalez for advice and T Wu for technical assistance with histochemistry, H. Shao for advice on reaggregation cultures, and J Waters, J. Kaye, and J Sprent for critical reading of the manuscript. Supported by a grant from the Lucille P. Markey Charitable Trust and by NIH grant AI32751 (W.L H.). W.L.H. is a Lucille P. Markey Scholar and R.B. is a fellow of the Strohm Inflammatory Bowel Disease Center at the Scripps Clinic and Research Foundation. W.H.F. was supported in part by the Foundation for Medical Research, Inc. This is manuscript 9491-IMM from the Scripps Research Institute.