PMC:17829 / 3474-6975 JSONTXT

{"project":"2_test","denotations":[{"id":"11178120-1380034-4390805","span":{"begin":114,"end":115},"obj":"1380034"},{"id":"11178120-1380034-4390806","span":{"begin":565,"end":566},"obj":"1380034"}],"text":"Methods:\nECs were isolated from human umbilical cord veins by digestion with collagenase as described previously [7] and then cultured. ECs in the third to fifth passage were used. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats of healthy blood donors by centrifugation over gradients of Ficoll-Hypaque. PBMCs were prepared immediately before starting the experiments.\nInteractions with ECs in PBMC populations were examined on hydrated bovine collagen gels in the wells of 16 mm macrowell tissue culture plates, as described previously [7]. To form a confluent monolayer on the collagen gels, 5 ×105 ECs per well were incubated overnight.\nTo measure monocyte interaction with ECs, PBMCs (3 ×106) were resuspended in fresh culture medium, layered on top of collagen gels with and without ECs and incubated at 37°C. The range of the incubation period was 15 minutes to 24 hours. Nonadherent cells (NAD) were harvested by washing twice with culture medium. Cells bound to the surface (BND) were enriched by washing each well twice with warm (37°C) Puck's EDTA, twice with warm (37°C) EGTA [0.5 mM EGTA in phosphate-buffered saline (PBS)] and once with cold (4°C) Puck's EDTA. Finally, for the recovery of those cells that had migrated into the collagen gels (MIG), 0.7 ml of a solution containing 0.1% collagenase, 1% (v/v) fetal calf serum and 50 mM Hepes buffer was added per well. The collagen gels were then minced gently with a pipette and incubated for 60 minutes at 37°C, after which the migrated PBMCs were removed by washing the wells twice with PBS. Each population (NAD, BND and MIG) was washed, resuspended in culture medium and counted under a microscope. In some experiments we studied monocyte migration into plain collagen gels. In these experiments no ECs were layered on the collagen gels; in other respects the experiments were performed exactly as described above.\nIn some experiments the EC monolayer was preactivated by incubation with TNF-α, IL-1α, macrophage inflammatory protein (MIP)-1α or interferon-γ (IFN-γ). To this end, the medium in each well was removed and the ECs were incubated for 5 hours at 37°C with or without the respective cytokines or chemokines (100 IU/ml TNF-α, IL-1α or IFN-γ, or 50 ng/ml MIP-1α). After this 5-hour pretreatment, each well was washed thoroughly and the migration assay was performed as described above.\nStaining of monocytes was performed with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD11a, CD33, CD45RA, CD45RO, CD49d (α4-integrin), CD54 (ICAM-1), CD86, HLA-DR, CD45RB and CD62L (L-selectin) (functions and ligands of these surface markers are shown in Table 1). To distinguish monocytes from other immune cells, all samples were counterstained with a phycoerythrin-labelled anti-CD14 monoclonal antibody. Cells (3 × 105 to 4 × 105 per sample) were incubated at 4°C for 30 minutes. Cells were then pelleted and resuspended in 250 μl of PBS before analysis was performed on a flow cytometer. All results are expressed as the respective mean fluorescence intensity among CD14-positive cells. Because not only monocytes but also ECs express CD54 (ICAM-1), in the analyses of the expression of CD54 on monocytes by fluorescence-activated cell sorting, monocytes were defined by both the scatter profile and the expression of CD14. In addition, ECs, which are considerably larger, were excluded by size.\nAll data are presented as means ± SD. Paired Student's t-tests were used for comparisons."}

{"project":"Colil","denotations":[{"id":"T37","span":{"begin":114,"end":115},"obj":"1380034"},{"id":"T38","span":{"begin":565,"end":566},"obj":"1380034"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Methods:\nECs were isolated from human umbilical cord veins by digestion with collagenase as described previously [7] and then cultured. ECs in the third to fifth passage were used. Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats of healthy blood donors by centrifugation over gradients of Ficoll-Hypaque. PBMCs were prepared immediately before starting the experiments.\nInteractions with ECs in PBMC populations were examined on hydrated bovine collagen gels in the wells of 16 mm macrowell tissue culture plates, as described previously [7]. To form a confluent monolayer on the collagen gels, 5 ×105 ECs per well were incubated overnight.\nTo measure monocyte interaction with ECs, PBMCs (3 ×106) were resuspended in fresh culture medium, layered on top of collagen gels with and without ECs and incubated at 37°C. The range of the incubation period was 15 minutes to 24 hours. Nonadherent cells (NAD) were harvested by washing twice with culture medium. Cells bound to the surface (BND) were enriched by washing each well twice with warm (37°C) Puck's EDTA, twice with warm (37°C) EGTA [0.5 mM EGTA in phosphate-buffered saline (PBS)] and once with cold (4°C) Puck's EDTA. Finally, for the recovery of those cells that had migrated into the collagen gels (MIG), 0.7 ml of a solution containing 0.1% collagenase, 1% (v/v) fetal calf serum and 50 mM Hepes buffer was added per well. The collagen gels were then minced gently with a pipette and incubated for 60 minutes at 37°C, after which the migrated PBMCs were removed by washing the wells twice with PBS. Each population (NAD, BND and MIG) was washed, resuspended in culture medium and counted under a microscope. In some experiments we studied monocyte migration into plain collagen gels. In these experiments no ECs were layered on the collagen gels; in other respects the experiments were performed exactly as described above.\nIn some experiments the EC monolayer was preactivated by incubation with TNF-α, IL-1α, macrophage inflammatory protein (MIP)-1α or interferon-γ (IFN-γ). To this end, the medium in each well was removed and the ECs were incubated for 5 hours at 37°C with or without the respective cytokines or chemokines (100 IU/ml TNF-α, IL-1α or IFN-γ, or 50 ng/ml MIP-1α). After this 5-hour pretreatment, each well was washed thoroughly and the migration assay was performed as described above.\nStaining of monocytes was performed with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD11a, CD33, CD45RA, CD45RO, CD49d (α4-integrin), CD54 (ICAM-1), CD86, HLA-DR, CD45RB and CD62L (L-selectin) (functions and ligands of these surface markers are shown in Table 1). To distinguish monocytes from other immune cells, all samples were counterstained with a phycoerythrin-labelled anti-CD14 monoclonal antibody. Cells (3 × 105 to 4 × 105 per sample) were incubated at 4°C for 30 minutes. Cells were then pelleted and resuspended in 250 μl of PBS before analysis was performed on a flow cytometer. All results are expressed as the respective mean fluorescence intensity among CD14-positive cells. Because not only monocytes but also ECs express CD54 (ICAM-1), in the analyses of the expression of CD54 on monocytes by fluorescence-activated cell sorting, monocytes were defined by both the scatter profile and the expression of CD14. In addition, ECs, which are considerably larger, were excluded by size.\nAll data are presented as means ± SD. Paired Student's t-tests were used for comparisons."}