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Volumn 282, Issue 5388, 1998, Pages 480-483

Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking

Author keywords

[No Author keywords available]

Indexed keywords

COLLAGEN; INTERLEUKIN 1; LATEX; LIPOPOLYSACCHARIDE; ZYMOSAN;

EID: 0032538522     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5388.480     Document Type: Review
Times cited : (703)

References (42)
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    • note
    • Endothelial cultures were established in 96-well plates and incubated at all times in Medium 199 supplemented with 20% heat-inactivated human serum. Transendothelial migration assays were conducted as described in (15-18). For some samples, during the preparation of polymerized collagen gels and before addition of endothelial cells, latex microspheres (0.65-μm diameter; Bangs Laboratories, Carmel, IN) or zymosan (ICN, Costa Mesa, CA; autoclaved in phosphate-buffered saline) was mixed with monomeric collagen at concentrations of 0.05% (w/v) or 0.0025% (w/v), respectively. LPS was undetectable in the collagen preparations, as tested with a limulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA). PBMCs were incubated with confluent, unstimulated endothelial monolayers for 1.5 hours and then washed to remove nonmigrated lymphocytes and monocytes (only about one-half of monocytes transmigrate into the collagen). Then Medium 199 containing 20% heat-inactivated human serum with or without soluble stimuli [recombinant human IL-1 (500 pg/ml), 30% MCM (14), or LPS (1 ng/ml)], when used, was added for 2 days.
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    • note
    • Reverse-transmigrated cells accumulated in the medium overlying endothelium and were pipetted off for collection. To obtain reverse-transmigrated cells that were loosely adherent to the apical surface of the endothelium, we also collected two subsequent washes in Hanks' buffered saline solution containing 1 mM EGTA.
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    • note
    • After collection of reverse-transmigrated cells, Hanks' buffered saline solution containing 1 mM EGTA and collagenase D (2 mg/ml) (Boehringer Mannheim) was added to each well and incubated at 37°C for 40 min. Solubilized cultures were transferred to a cell strainer and washed with medium to obtain cell suspensions devoid of collagenous debris. Digests contained a mixture of monocyte-derived and endothelial cells. Cell viability was > 95%. In control experiments, treatment of reverse-transmigrated cells with collagenase by means of this procedure did not alter the phenotypic properties of these cells.
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    • note
    • + sorted cells to a level of 10% of the total population to match their abundance in nonsorted cells and used in reverse transmigration assays. Lymphocytes that were not depleted by magnetic beads were discarded during the cell sorting by setting a gate to remove cells with the forward and side scatter properties of lymphocytes.
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    • note
    • Portions of cells were incubated on ice with mAbs (1 μg/ml) to the following antigens: CD14 (3C10), CD32 (IV.3; Mederex, Annandale, NJ), CD64 (10.1; Ancell), CD80 (P1.H5.A1; Ancell), CD83 (HB15a; Immunotech, Miami, FL), and CD86 (IT2.2; Pharmingen). Nonbinding control mAbs included immunoglobulin G1 (IgG1) and IgG2a control from Immunotech, UPC10 (Sigma), and MOPC 21 (Sigma). These mAbs were detected with FITC-conjugated goat antibody to mouse IgG (Dako). R-phycoerythrin-conjugated antibody to HLA-DR (Becton-Dickinson) was used for two-color flow cytometry. Samples were analyzed in a FACSCalibur instrument, with CellQuest software.
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    • note
    • Cytospins of reverse-transmigrated cells and subendothelial cells were stained with isotype-matched control mAb UPC 10 (Sigma), p55 mAb, or CD68 mAb followed by horseradish peroxidase-conjugated antibody and diaminobenzamidine substrate. Nuclei were counterstained with hematoxylin.
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    • note
    • E-rosette-positive T cells were prepared from human blood and further purified by panning with HLA-DR mAb.
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    • note
    • We thank R. Liebman for isolation and culture of endothelial cells. This work was funded by grants HL46849 (W.A.M.), AI13013 (R.M.S.), and AI40045 (R.M.S.) from the NIH. G.J.R. was supported by National Research Service Award HL09722. S.B. was the recipient of a fellowship from the National Health and Research Development Program, AIDS, Health and Welfare, Canada. W.A.M. is an Established Investigator of the American Heart Association.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.