Essay on Immunofluorescence Report

Bio 385-01

Immunofluorescence Microscopy of SNB-19 Glioblastoma Cell Cultures using Monoclonal Antibodies and Colcemid Treated Plates

I. Introduction Immunofluorescence microscopy is used commonly in the fields of cell biology and microbiology. It is a useful technique to ascertain a visualization of a particular intracellular protein of the experimenter’s choosing by taking advantage of the antigen-antibody response and tagging an antibody with a fluorescent marker. One reason why this technique is commonly used is because the effects of certain drugs on intracellular structures can be detected, such as the components of the cytoskeleton. For instance, a group of researchers took a look at the effects of curcumin, a drug derived from the root of the Curcuma longa plant, on microtubule structures in three cancer cell lines (HT-29, Caco-2, and MCF-7) and Plasmodium falciparum (human malaria parasite)(1) (Chakrabarti et al, 2013). This team of researchers found that curcumin functioned similarly to colchicine in that it most likely bound to the site at which α and ß-tubulin interface. Colcemid is another drug that has a similar function to that of colchicine and curcumin in that it can disrupt the ability to disrupt microtubule formation, which could cause problems during anaphase of the mitotic cycle. The cytoskeleton is a filamentous protein structure of a eukaryotic cell, which allows the cell to maintain and change shape, as well as transfer materials from one pole of the cell to the other via motor protein action. Microtubules, microfilaments, and intermediate filaments are the major components that make up the eukaryotic cytoskeleton. The microtubules, which are the largest in diameter (25 nm) and are polymerized from the negative α-tubulin end to the positive ß-tubulin end (Elliot, 2014)(2), are composed of 13 α and ß-tubulin protomers (combination of α and ß-tubulin monomers). They are responsible for polarization of organelles, structural integrity, transport of materials, as well as attachment to the kintechore of chromosomes (anaphase). Interactions between antigens and antibodies were also highlighted as a major part of immunofluorescence microscopy. Antibodies that are commonly used are either characterized as monoclonal or polyclonal. Monoclonal antibodies recognize a singular epitope or binding region on the antigen of interest, while polyclonal antibodies can recognize multiple epitopes on the antigen of interest (Elliot, 2014)(3). Because of this functionality, monoclonal antibodies are preferred in a lab setting for the purposes of efficient isolation for an antigen of interest. Typically, the antibody is fluorescently tagged with a fluorochrome, such as FITC (Fluoresceine-isothiocyanate), for visualization of an antigen of interest (e.g. anti-tubulin, localized around the nucleus). For the experiment, human glioblastoma cells from cell line SNB-19 were cultured on regular nutrient media and colcemid treated plates. They were then exposed to a primary (mouse anti-tubulin (monoclonal) to recognize protein of interest) and secondary (containing FITC tag and targeting primary antibody) antibody in order to visualize the protein of interest, which is α -tubulin, via immunofluorescence microscopy. The objective of the experiment is to visualize the effects that the drug, colcemid, has on microtubule polymerization. Based on the information provided about colchicine and curcumin, it was most likely that colcemid would cause inhibition of anti-tubulin polymerization.

II. Methods Some SNB-19 human glioblastoma cells were seeded into 2 35-millimeter plates; one of which was pretreated colcemid. The plates were washed using Tris Buffer Solution (a.k.a. TBS) after the plates were exposed to different solutions and antibodies in subsequent steps of the experiment. The medium that was originally on the plate was removed, and then the plate was washed with 2 mL of TBS. Subsequently, 5% glacial acetic acid in

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