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ICAM-1 protein levels in the supernatant were determined by an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) that recognizes all splice variants from 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA) and were normalized to total protein (Bio-Rad, Hercules, CA).
The following sense and antisense sequences were used to target murine ICAM-1: sense, 5′-ACC GAT CGT CAC GGC GAT TTA TAA GTT CTC TAT AAA TCG CCG TGA CGA TCT TTT TC-3′; antisense, 5′-TGA AGA AAA AGA TCG TCA CGG CGA TTT ATA GAG AAC TTA TAA ATC GCC ACG AT-3′.
ICAM-1 upregulation was induced by retinal laser photocoagulation and streptozotocin (STZ).
After the administration of GFP expression plasmid with HT and IV, histologic analysis showed GFP fluorescence in every layer of the murine retina.
Green fluorescent protein (GFP) expression plasmid vector is used as a transfection marker in the retinal cells.
ICAM-1 expression was analyzed by enzyme-linked immunosorbent assay and flow cytometry.
The authors describe the application of si RNA to suppress ICAM-1 expression on the murine neurosensory retina or retinal pigment epithelial (RPE) cells using a hydrodynamics-based transfection technique (HT) and intravitreal injection (IV) in vivo.
ICAM-1-specific plasmid si RNAs designed from the murine gene sequence were transfected into the retina using HT and IV in vivo.Si RNA expression mediated by this plasmid causes efficient and specific downregulation of ICAM-1 expression, suggesting that it can be silenced by plasmid si RNA in murine retina in vivo.This technology may lead to novel concepts to reduce retinal neovascular disease by inhibiting leukocyte infiltration. The mechanism by which small-interfering RNAs (si RNAs) recognize and bind to their target RNA in mammalian cells and suppress target gene expression posttranscriptionally is assumed to include base-specific recognition.Leukocytes play a critical role in ocular diseases such as uveitis, diabetic retinopathy, and choroidal neovascularization.Intercellular adhesion molecule (ICAM)-1 is essential for the migration of leukocytes.Naked RNA cannot penetrate cellular lipid membranes, and systemic application of unmodified si RNA is unlikely to be successful for resolving these problems and delivering a higher level of si RNA and release for a long period.