Date Presented

Spring 5-18-2024

Document Type

Thesis

First Advisor

Dr. David Freier

Second Advisor

Dr. Price Blair

Third Advisor

Dr. Christine Terry

Abstract

The RAW 264.7 murine macrophage is a prevalent cell line in mammalian cell culture and is commonly used for the study of inflammation The standard medium for its growth and maintenance is Dulbecco's Modified Eagle's Medium (DMEM), but in a portion of published work, RPMI 1640 is substituted. RPMI 1640 lacks the high level of glucose present in DMEM (4.5 g/L). The purpose of this work is to compare the function of RAW 264.7 cells grown and maintained in DMEM versus RPMI 1640. Both media are supplemented with 10% fetal bovine serum, sodium bicarbonate, and 100 IU Penicillin Streptomycin 100μg. DMEM includes the addition of 4.5 g/L glucose. Following a dose response exposure of RAW 264.7 to a range of 0 ng/mL to 100 ng/mL lipopolysaccharide (LPS) from E. coli (serotype O55:B5) for 24 hours in a 5%CO2/ 37°C environment, nitrite production was measured using the Griess reaction. Initial results suggest RAW 264.7 cells grown in RPMI 1640 have a decreased nitric oxide response. Once the difference between DMEM and RPMI 1640 was established, glucose was added to RPMI 1640 to recover the nitric oxide response levels observed in DMEM cultures, and cells grown in high glucose RPMI 1640 did have similar responses to DMEM cells. Variance in inflammatory responses between cells in different growth media validates the need for enhanced cell culture literature and standardization.

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