Abstract
Fibroblast growth factors (FGFs) are part of a large family of polypeptide growth factors that play a large role in the development and regulation of organisms using intracrine, paracrine, and endocrine cell signaling mechanisms. FGFs are known to be found in both vertebrate and invertebrate organisms. Most recently they have also been identified in viruses, such as baculoviruses. A sequence analysis of the baculovirus genomes has found that they encode for a viral fibroblast growth factor homolog (vfgf) early on in their stage of infection (Katsuma et al. 2006). Although there have been studies from other labs that have revealed a great deal about vFGF function, to date, nothing has been published on the potential mitogenic activity of vFGFs. Dr. Finnerty and earlier research students have obtained promising results, showing vFGFs from two baculoviruses to be mitogenic, but these results needed to be reproduced and replicated in multiple cell lines to have sufficient data for publication. To test the effects of vFGFs on cell mitogenic activity, the best method to assess cell viability and cell proliferation needed to be used, which is colorimetric assays. My research focused on finding the most accurate in-vitro colorimetric assay that measures cell proliferation with insect SF9 cells and mammalian NIH/3T3 cells. Such assays included the Resazurin assay, the Crystal Violet assay, and the CCK-8 assay. To accurately measure cell proliferation, I used a multi-well plate reader that measured the dyes' absorbance at specific wavelengths varying from 450nm to 590nm, depending on the reagent. From the data collected, I created a variety of standard curve graphs and produced figures that compared each reagent's sensitivity. The slopes of the Resazurin assay graphs all show a trend of starting positive with the smaller seeding densities and then turning negative with the larger densities, which is not ideal for future experiments. The CCK-8 assay graphs all show a trend of positive slopes with both the small and large seeding densities for each cell line, while the crystal violet assay graphs showed similar trends as Resazurin. I used 96-well plates to have various replicates of each cell concentration and compared the confidence interval ranges for each assay. The assay with the lowest confidence intervals throughout all the graphs was CCK-8. The last component I used to choose the best in-vitro colorimetric assay was the correlation coefficients in each graph. Out of all three reagents, CCK-8 had the best coefficients in the standard curve graphs, with values above 0.9. Therefore, the CCK-8 assay was found to be the best in-vitro colorimetric assay to measure cell proliferation with both SF9 and NIH/3T3 cells. The CCK-8 standard curve graphs I created for each cell line will also help as a template to measure the mitogenic activity of cells after adding vFGF for future experiments.
College
College of Science & Engineering
Department
Biology
Campus
Winona
First Advisor/Mentor
Casey Finnerty
Start Date
4-19-2023 9:00 AM
End Date
4-19-2023 10:00 AM
Presentation Type
Poster Session
Format of Presentation or Performance
In-Person
Session
1a=9am-10am
Poster Number
22
Included in
Comparison of Colorimetric Assays to Use for the Investigation of the Mitogenic Activity of vFGF in Cell Cultures
Fibroblast growth factors (FGFs) are part of a large family of polypeptide growth factors that play a large role in the development and regulation of organisms using intracrine, paracrine, and endocrine cell signaling mechanisms. FGFs are known to be found in both vertebrate and invertebrate organisms. Most recently they have also been identified in viruses, such as baculoviruses. A sequence analysis of the baculovirus genomes has found that they encode for a viral fibroblast growth factor homolog (vfgf) early on in their stage of infection (Katsuma et al. 2006). Although there have been studies from other labs that have revealed a great deal about vFGF function, to date, nothing has been published on the potential mitogenic activity of vFGFs. Dr. Finnerty and earlier research students have obtained promising results, showing vFGFs from two baculoviruses to be mitogenic, but these results needed to be reproduced and replicated in multiple cell lines to have sufficient data for publication. To test the effects of vFGFs on cell mitogenic activity, the best method to assess cell viability and cell proliferation needed to be used, which is colorimetric assays. My research focused on finding the most accurate in-vitro colorimetric assay that measures cell proliferation with insect SF9 cells and mammalian NIH/3T3 cells. Such assays included the Resazurin assay, the Crystal Violet assay, and the CCK-8 assay. To accurately measure cell proliferation, I used a multi-well plate reader that measured the dyes' absorbance at specific wavelengths varying from 450nm to 590nm, depending on the reagent. From the data collected, I created a variety of standard curve graphs and produced figures that compared each reagent's sensitivity. The slopes of the Resazurin assay graphs all show a trend of starting positive with the smaller seeding densities and then turning negative with the larger densities, which is not ideal for future experiments. The CCK-8 assay graphs all show a trend of positive slopes with both the small and large seeding densities for each cell line, while the crystal violet assay graphs showed similar trends as Resazurin. I used 96-well plates to have various replicates of each cell concentration and compared the confidence interval ranges for each assay. The assay with the lowest confidence intervals throughout all the graphs was CCK-8. The last component I used to choose the best in-vitro colorimetric assay was the correlation coefficients in each graph. Out of all three reagents, CCK-8 had the best coefficients in the standard curve graphs, with values above 0.9. Therefore, the CCK-8 assay was found to be the best in-vitro colorimetric assay to measure cell proliferation with both SF9 and NIH/3T3 cells. The CCK-8 standard curve graphs I created for each cell line will also help as a template to measure the mitogenic activity of cells after adding vFGF for future experiments.
