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HPB-ALL cells were plated on VCAM-1 in the temperature controlled environment of an inverted Nikon (Melville, NY) Diaphot-TMD microscope for 1 hour.
Figure 6demonstrates HPB-ALL spreading on VCAM-1 over a period of 1 hour, where images from the first 10 minutes were obtained from recorded videotape, followed by a 60 minute time point.
Previous reports have demonstrated that ADP-ribosylation of B-lymphoid cell lines by C3 treatment inhibits phorbol ester induced cellular homotypic aggregation [ 18 ] . Also, phorbol ester and chemoattractant (IL-8 and fMLP) induced pre-B lymphoblastoid adhesion to VCAM-1 and neutrophil adhesion to fibrinogen was abrogated in a dose-dependent manner by C3 exoenzyme [ 36 ] . This may indicate that in cells of hematopoietic lineages, the induction of an increased adhesive state may be susceptible to regulation by Rho proteins.
Monoclonal antibodies were purchased from commercial vendors unless otherwise stated: CD3, IgG1; CD68, IgG1; CD34, IgG1; CD45, IgG1, D105, IgG1; and IgG2a controls (Dako Corporation, Carpinteria, CA, USA); anti-HLA-DR, IgG2a; CD14, IgG2b; CD34, IgG1 (Becton Dickinson, San Jose, CA, USA); anti-vimentin, IgG1; IgG2b control (Serotec, Kidlington, Oxfordshire, UK); anti-VCAM-1, IgG1; anti-αvβ3 (vitronectin receptor), IgG1 (Pharmingen, San Diego, CA, USA); anti-collagen-type-1, IgG1 (Sigma Diagnostics); anti-osteocalcin, IgG1 (ABOC-5021, Haematologic Technologies, Essex Junction, VT, USA) anti-IgG1.
When HPB-ALL cells are plated onto β1 integrin substrate such as fibronectin, vascular cell adhesion molecule-1 (VCAM-1), or even antibodies to α4β1 integrins, they respond by extending pseudopodia and displaying a highly flattened, spread morphology (figure 3, panels 4, 7, and 10, and ref [ 32 ] ). However, electroporation of C3 appeared to alter HPB-ALL morphology resulting in the extension of very fine branched finger-like cellular processes when cells were plated on the β1 integrin substrates Fn (figure 3, panel 6), VCAM-1 (figure 3, panel 9), or a mAb specific for β1 integrins (figure 3, panel 12).
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