Location
Room 232, Schewel Hall
Access Type
Campus Access Only
Entry Number
79
Start Date
4-5-2023 9:30 AM
End Date
4-5-2023 9:45 AM
College
College of Medical Sciences
Department
Biomedical Science
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 Bone Marrow Derived Macrophages (BMDM), reducing 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 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 removal of supernatant for quantification of nitrites in solution by the Griess assay. To correct for growth rate differences of BMDM compared to RAW 264.7 and J774A.1 cells when measuring nitrite response levels, cells will be dislodged (using 0.5 mM EDTA in phosphate buffered saline), counted by hemacytometer, and normalized (per 10,000 cells). Correcting the difference in macrophage growth rates should allow for a clearer comparison of inflammatory responses among the three types of macrophages.
Faculty Mentor(s)
Dr. David Freier Dr. John Strysky Dr. Blair Price
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A Functional Comparison of Murine Bone Marrow Derived Macrophages to RAW 264.7 and J774A.1 Cells
Room 232, Schewel Hall
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 Bone Marrow Derived Macrophages (BMDM), reducing 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 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 removal of supernatant for quantification of nitrites in solution by the Griess assay. To correct for growth rate differences of BMDM compared to RAW 264.7 and J774A.1 cells when measuring nitrite response levels, cells will be dislodged (using 0.5 mM EDTA in phosphate buffered saline), counted by hemacytometer, and normalized (per 10,000 cells). Correcting the difference in macrophage growth rates should allow for a clearer comparison of inflammatory responses among the three types of macrophages.