Date Presented

Spring 5-1-2023

Document Type

Thesis

Department

Biomedical Science

First Advisor

David O. Freier, MA, PhD

Second Advisor

Price Blair, PhD

Third Advisor

John Styrsky, PhD

Abstract

Parallel examination of bone marrow-derived macrophages (BMDM) to the viral transformed murine RAW 264.7 and J774A.1 cell lines provides a basis for comparison for experimental models of inflammation. Equivalent inflammatory responses would allow the substitution of the RAW 264.7 or J774A.1 cells for BMDM, reducing the use of animals in experimentation. The mammalian cell lines are maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) complete with passage by scraping every 72 hours. BMDM are generated by culturing bone marrow cells from femurs of female Swiss mice (Hilltop Lab Animals, Inc.) with murine macrophage colony-stimulating factor for 7 days. The two cell lines, RAW 264.7 and J774A.1, and BMDM are simultaneously seeded at 4x10^5 cells per well in triplicate in 500μL of DMEM complete, then incubated for 14 hours. Attached cells are then washed two times with DMEM before stimulation with lipopolysaccharide (LPS) from E. coli (O55:B5) to induce an inflammatory response. A dose-response exposure to 0, 1, 10, and 100 ng/mL of LPS for 24 hours is followed by removing the supernatant to quantify nitrites in solution by the Griess assay. To correct for a growth rate difference of BMDM compared to RAW 264.7 and J774A.1 cells when measuring nitrite response levels, cells were dislodged using 0.5 mM EDTA in phosphate-buffered saline, counted by hemacytometer, and normalized per 10,000 cells. Once correcting the difference in macrophage growth rates a clearer comparison of inflammatory responses among the three types of macrophages was able to be drawn.

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