Analysis of Phagocytic and Cytotoxic Activity in Macrophage
In the Lab 3 module we will analyze both the phagocytic and cytotoxic activity of macrophages. By doing so, we will gain an understanding of how macrophages function in the innate immune response and how they respond when co-cultured with tumor cells after stimulation with a lipid mediator. In lab #3-A we will analyze the latter of these responses, or the phagocytic activity of macrophages. This will be done by isolating macrophages from the intra-peritoneal (IP) cavity and adding latex beads, instead of bacteria, to act as a substrate for the macrophages. After incubation, the macrophages will be visualized using Wright’s stain to determine which beads have adhered to or have been engulfed by the macrophages. In lab #3-B, the cytotoxicity of macrophages will be analyzed by incubating, or co-culturing, a macrophage cell line with a mouse mastocytoma tumor cell line. The cytotoxicity of the macrophages will be based on their ability to kill the tumor cells. This will be assayed by measuring the release of an enzyme, lactate dehydrogenase (LDH), from the tumor cells as a colorimetric reaction. Lastly, we will use the CytoTox 96 Non-Radioactive Cytotoxicity Assay to perform colorimetric analysis to determine the concentration of LDH in a solution with the aid of a coloring reagent (1).
Innate immunity is the body’s natural and primary resistance to infectious disease and involves the recognition of foreign pathogens, followed by a response to eliminate the pathogens. The common barriers that comprise the innate immune response include: the skin and mucous membranes (anatomic); body temperature, stomach pH, and various soluble factors such as lysozymes in tears (physiological); uptake of extracellular material by a cell (endocytic); and the ingestion of microorganisms and particulate material from the extracellular environment by expansion of the cellular membrane...