Abstract
The epithelial cell is usually the first host cell that interacts with the microbiota present at mucosal surfaces. Although initially thought of as “bystander” cells with barrier function, the epithelial cell is now known to be a sentinel cell in the recognition and discrimination of commensal and pathogenic microorganisms and a key cell in initiating subsequent innate and adaptive immune responses. Here, we describe the main assays utilized in analyzing the activation of epithelial cell signaling (western blotting), transcription factors (TransAm), gene expression (quantitative reverse transcription PCR (qRT-PCR)), cytokine responses (ELISA, Luminex), and damage induction (lactate dehydrogenase (LDH) release). While our laboratory focuses on the epithelial response to Candida pathogens, these assays can be applied universally to analyze the activation of epithelial cells in response to any microbial pathogen.
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References
Renart J, Reiser J, Stark GR (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A 76:3116–3120
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354
VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626
Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51:2415–2418
Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874
Schmidt SD, Mazzella MJ, Nixon RA, Mathews PM (2012) Abeta measurement by enzyme-linked immunosorbent assay. Methods Mol Biol 849:507–527
Avrameas S (1969) Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6:43–52
Van Weemen BK, Schuurs AH (1971) Immunoassay using antigen-enzyme conjugates. FEBS Lett 15:232–236
Maes M, Vanhaecke T, Cogliati B, Yanguas SC, Willebrords J, Rogiers V, Vinken M (2015) Measurement of apoptotic and necrotic cell death in primary hepatocyte cultures. Methods Mol Biol 1250:349–361
Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70
Smith SM, Wunder MB, Norris DA, Shellman YG (2011) A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time. PLoS One 6:e26908
Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA (2015) A novel method for assessment of natural killer cell cytotoxicity using image cytometry. PLoS One 10:e0141074
Ngoka LC (2008) Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci 6:30
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408
Shapiro AL, Vinuela E, Maizel JV Jr (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–820
Acknowledgments
This work was supported by grants from the Medical Research Council (MR/M011372/1), Biotechnology and Biological Sciences Research Council (BB/N014677/1), National Institutes of Health (R37-DE022550), King’s Health Partners Challenge Fund (R170501), the Rosetrees Trust (M680), and the NIH Research at Guys and St. Thomas’s NHS Foundation Trust and the King’s College London Biomedical Research Centre (IS-BRC-1215-20006).
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Nikou, S. et al. (2021). Analysis of Epithelial Cell Responses to Microbial Pathogens. In: Bignell, E. (eds) Host-Fungal Interactions. Methods in Molecular Biology, vol 2260. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1182-1_5
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DOI: https://doi.org/10.1007/978-1-0716-1182-1_5
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