Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures |
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Authors: | Kathleen M. McNeeley Zhe Sun Susan T. Sharfstein |
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Affiliation: | (1) Department of Bioengineering, University of Toledo, 43606 Toledo, OH, USA;(2) Present address: Department of Biomedical Engineering, Georgia Institute of Technology, 30332-0535 Atlanta, GA, USA;(3) Present address: Department of Medical Physiology, Texas A&M University System Health Science Center, 77843 College Station, TX, USA;(4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Ricketts Building, 110 8th Street, 12180-3590 Troy, NY, USA |
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Abstract: | Flow cytometry was used to evaluate the effects of hyperosmotic stress on cell-cycle distribution and cell-associated immunoglobulins for murine hybridoma cells grown in batch culture. Paraformaldehyde/methanol fixation substantially increased the fluorescence signal for intracellular immunoglobulins compared to ethanol fixation. For surface immunoglobulins, similar fluorescence signals were observed regardless of fixation method. Dual staining of immunoglobulins and cellular DNA was employed to determine immunoglobulin pool size as a function of cell-cycle phase. The intracellular immunoglobulin pool sizes increased as the cells progressed through the cell cycle for both control and hyperosmotic cultures. For control cultures, the immunoglobulin pool size increased during the exponential phase of culture, followed by a decrease as the cultures entered stationary phase. In contrast, hyperosmotic cultures showed an initial decrease in immunoglobulin pool size upon the application of osmotic shock, followed by an increase to a level above that of control cultures. This behavior was observed in all phases of the cell cycle. In addition, hyperosmotic cultures exhibited an increase in cell size when compared to control cultures. When normalized for cell size, the intracellular immunoglobulin concentration in hyperosmotic cultures was initially lower than in control cultures and subsequently increased to slightly above the level of control cells. Cells in all phases of the cell cycle behaved in a similar manner. There was no apparent relationship between the intracellular antibody concentration and the rate of antibody secretion. |
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Keywords: | Cell cycle Flow cytometry Hybridoma Hyperosmotic stress Immunoglobulin Monoclonal antibody |
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