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Ankit's Paper - Examination of Differentiated and...

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Examination of Differentiated and Non-Differentiated Cells Ankit Patel Joanne Manaster/Eric Salm Labs 5-8 April 28, 2009
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INTRODUCTION In our lab we will be taking a closer look at differentiated and non-differentiated 3T3-LI cells to observe which proteins are present in which. We believe, the differentiated cells will have different proteins than un-differentiated because many proteins will need to be turned on during the process of differentiation, or carry on cell’s specific tasks. For instance, myoD1, is responsible for differentiation of myoblasts into mature muscle cells. These proteins will allow them to transform and carry out their specific tasks. As first, we differentiated fibroblasts into adipocytes. This portion of the lab will examine the properties of stem cells. Stems cell have the capability of self-renewing and multilineage differentiation. In vivo, they are capable of functional reconstitution of a given tissue. Fibroblasts are able to transform into any of the connective tissue cells. In our lab, we will differentiate fibroblast into adipocytes. Adipocytes, when removed from the body and cultured in presence of de-differentiating factors can revert back to fibroblast and then be forced to re-differentiate via external factors into osteoblasts (Manaster 51). Thus, fibroblasts are considered to be plastic. We can transform fibroblasts by regulating composition of the extracellular matrix (ECM), cell shape, hormones and growth factors. During this portion, we will stain adipocytes –which have been transformed by 3T3-L1s- with Oil Red O (a fat soluble dye) for triglycerides. By staining with Oil Red O, we will be able to analyze the cells with respect to their difference in shape from fibroblast. We will then work with C2C12 cells. These cells demonstrate the concept of spontaneous differentiation in response to cellular contact. As these cells grow and become more confluent, they will begin to fuse with each other, it will trigger genetic changes and the production of several proteins known to be found in mature skeletal muscles. Then using our cells, we will liberate the proteins from our cells. We will use these to determine which proteins our differentiated cells produce and which the non-differentiated cells don’t. We will mechanically destroy all plasma membranes by scraping and sonication of cells to obtain the proteins. Using the lysates of our proteins, we will run a SDS-PAGE for myoblasts, myotubes, fibroblasts and adipocytes. SDS is an ionic detergent that disrupts membranes by intercalating into phospholipids bilayer and solubilizing lipids and proteins; and since SDS is negatively charged, it will confer its net negative charge to the proteins. Beta-mercaptoethanol, will disrupt disulfide bonds to make proteins linearized (Blinstrup 24). This is important step because polyacrylamide gel electrophoresis (PAGE) will separate proteins by their size.
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