Poster Session
Location
Memorial Ballroom, Hall Campus Center
Access Type
Open Access
Entry Number
25
Start Date
4-10-2019 12:00 PM
End Date
4-10-2019 1:15 PM
College
Lynchburg College of Arts and Sciences
Department
Biology
Abstract
The purpose of this experiment is to develop comparative methodology for analyzing RAW 264.7 murine macrophage responses to bacterial lipopolysaccharide (LPS). These results will yield two endpoints relative to nitric oxide production: nitrites in solution from the Greiss assay, and protein expression of inducible nitric oxide synthase (iNOS). The RAW 264.7 cell line is a standard culture model of macrophage activity and stimulation with LPS induces inflammatory reactions. This process has evolved to kill ingested pathogens and activate local immune responses, including the production of nitric oxide, synthesized by iNOS. RAW 264.7 cells are stimulated with LPS doses from 1 – 100 ng/mL for 24-hours. Nitrite, a breakdown product of nitric oxide, is measured by the Greiss reaction and spectrophotometry to determine cell activity. The cells from the LPS Dose Response experiment are then treated with a RIPA mixture which consequently degrades the cells, leaving the proteins intact. These isolated proteins are stored in a freezer for later use in protein analysis. Western blot analysis of iNOS expression will be performed using the LiCor Odyssey system. These results will be cross-referenced with those of the LPS dose response test in order to correlate nitric oxide production and the activity of iNOS.
Faculty Mentor(s)
Dr. David Freier
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Included in
Comparative Methodology for Analyzing Nitric Oxide Production in RAW 264.7 Murine Macrophages in Response to Lipopolysaccharide Treatment
Memorial Ballroom, Hall Campus Center
The purpose of this experiment is to develop comparative methodology for analyzing RAW 264.7 murine macrophage responses to bacterial lipopolysaccharide (LPS). These results will yield two endpoints relative to nitric oxide production: nitrites in solution from the Greiss assay, and protein expression of inducible nitric oxide synthase (iNOS). The RAW 264.7 cell line is a standard culture model of macrophage activity and stimulation with LPS induces inflammatory reactions. This process has evolved to kill ingested pathogens and activate local immune responses, including the production of nitric oxide, synthesized by iNOS. RAW 264.7 cells are stimulated with LPS doses from 1 – 100 ng/mL for 24-hours. Nitrite, a breakdown product of nitric oxide, is measured by the Greiss reaction and spectrophotometry to determine cell activity. The cells from the LPS Dose Response experiment are then treated with a RIPA mixture which consequently degrades the cells, leaving the proteins intact. These isolated proteins are stored in a freezer for later use in protein analysis. Western blot analysis of iNOS expression will be performed using the LiCor Odyssey system. These results will be cross-referenced with those of the LPS dose response test in order to correlate nitric oxide production and the activity of iNOS.