Frye_and_Edidin_to_post

Frye_and_Edidin_to_post - Frye and Edidin Rapid intermixing...

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Frye and Edidin: Rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. A. Major questions: 1. How do cell membranes change shape quickly as cell morphology changes? (e.g., during phagocytosis or cell migration) 2. What is the nature of the cell membrane? 3. Is the membrane fluid or rigid? B. Hypothesis : The membrane is fluid. Prediction: A fluid membrane will allow membrane components (proteins) to freely diffuse laterally in the membrane.
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C. Approach: Cell Biology Experimental Strategy:
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D. Tools required for these experiments 1. Cells: •Cells taken directly from an organism will proliferate for many generations but most of the cells stop dividing after about 50 generations. •To maintain cells in culture requires that the cells be immortalized so that they can proliferate almost indefinitely rather than dying out. We often call these immortalized cells a “cell line”. •For this study: •Mouse cell line, cIID •immortalized fibroblast cells have acquired genetic mutations •Human cell line, VA-2 •SV40 transformed line •The viral proteins alter cell cycle regulation to maintain proliferation. 2. Method to fuse cells: Inactivated Sendai virus •Fusogen •Why and how? •Inactivated to prevent actual viral infection •Purified F or fusion protein from this virus will induce cell fusion
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Protocol •Chill cells •incubate with primary antibody (MBOC 23-14). •wash •incubate with secondary antibody •wash D. Tools required for these experiments 3. Method to visualize mixing of cell surface proteins: Indirect immunofluorescence •secondary antibody is tagged using fluorescence to allow detection •Note that because the antibodies are binding proteins exposed on the surface of the cell, there is no need to fix and permeabilize the cells. The cells are alive at the time of viewing.
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Alloantiserum •Immunize a mouse strain with spleen and lymph cells of another strain. •Elicits "Alloantiserum" -reacts against histocompatibility antigens (MHC molecules).
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This note was uploaded on 10/19/2009 for the course B M B 460 taught by Professor Hanna-rose,w during the Spring '08 term at Penn State.

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Frye_and_Edidin_to_post - Frye and Edidin Rapid intermixing...

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