Dose Response Effect of Perfluorooctanoic Acid on Inflammatory Responses in RAW 264.7 Murine Macrophages
Location
Room 232, Schewel Hall
Access Type
Campus Access Only
Entry Number
80
Start Date
4-5-2023 8:00 AM
End Date
4-5-2023 8:15 AM
College
Lynchburg College of Arts and Sciences
Department
Biomedical Science
Keywords
RAW 264.7 Murine Macrophages, Perfluorooctanoic acid (PFOA)
Abstract
PFOA, or Perfluorooctanoic acid, is a common pollutant found in consumer products such as non-stick cookware or flame-retardant fabrics. Numerous ex vivo studies indicate PFOA alters antibody levels, decreases the number of lymphocytes and macrophages by inducing apoptosis, and increases the likelihood of developing cancer and autoimmunity. Despite the abundance of literature, no clear mechanism of immunotoxicity has yet to be identified. Functional changes to B and T cell responses are directly connected to PFOA intoxication, but modulation of innate responses including macrophages are not as well characterized. In RAW 264.7 Murine Macrophages, reports indicate PFOA increases measures of inflammation including the production of pro-inflammatory cytokines such as IL-6, IL-10, and TNF-alpha. Mouse macrophage cell model, RAW 264.7 cells, were cultured in complete DMEM and incubated at 4 x10^5 cells/well in a 24 well plate (500 μl volume of DMEM) overnight. Cells were then pretreated with PFOS (Sigma Chemical CAS no. 2795-39-3) at 10uM, 20 uM, 100 uM, and 200 uM doses for 24 hours. LPS from E.coli O55:B5 (Sigma Chemical) at 100 ng/ml was later added to examine the response of PFOA stimulate macrophages. Samples were collected after 24 hrs to measure the amounts of pro-inflammatory cytokines by ELISA. A Griess Assay was performed on 50uL samples from each well to determine level of nitrite in solution as an additional measure of inflammatory response. To verify the results of the cell culture, a Neural Network was contructed using PyTorch as well as data from Drugbank and Pubchem.
Faculty Mentor(s)
Dr. David Freier
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Dose Response Effect of Perfluorooctanoic Acid on Inflammatory Responses in RAW 264.7 Murine Macrophages
Room 232, Schewel Hall
PFOA, or Perfluorooctanoic acid, is a common pollutant found in consumer products such as non-stick cookware or flame-retardant fabrics. Numerous ex vivo studies indicate PFOA alters antibody levels, decreases the number of lymphocytes and macrophages by inducing apoptosis, and increases the likelihood of developing cancer and autoimmunity. Despite the abundance of literature, no clear mechanism of immunotoxicity has yet to be identified. Functional changes to B and T cell responses are directly connected to PFOA intoxication, but modulation of innate responses including macrophages are not as well characterized. In RAW 264.7 Murine Macrophages, reports indicate PFOA increases measures of inflammation including the production of pro-inflammatory cytokines such as IL-6, IL-10, and TNF-alpha. Mouse macrophage cell model, RAW 264.7 cells, were cultured in complete DMEM and incubated at 4 x10^5 cells/well in a 24 well plate (500 μl volume of DMEM) overnight. Cells were then pretreated with PFOS (Sigma Chemical CAS no. 2795-39-3) at 10uM, 20 uM, 100 uM, and 200 uM doses for 24 hours. LPS from E.coli O55:B5 (Sigma Chemical) at 100 ng/ml was later added to examine the response of PFOA stimulate macrophages. Samples were collected after 24 hrs to measure the amounts of pro-inflammatory cytokines by ELISA. A Griess Assay was performed on 50uL samples from each well to determine level of nitrite in solution as an additional measure of inflammatory response. To verify the results of the cell culture, a Neural Network was contructed using PyTorch as well as data from Drugbank and Pubchem.