Impact of Different Cell Isolation Techniques on Lymphocyte Viability and Function A. B. Klein, S. G. Witonsky, S. Ansar Ahmed, S. D. Holladay, and R. M. Gogal, Jr. Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, USA L. Link Department of Biology, Virginia Tech, Blacksburg, Virginia, USA C. M. Reilly Department of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Blacksburg, Virginia, USA Abstract: The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte pro- liferation was measured using the Alamar Blue TM assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant Address correspondence to R. M. Gogal, Jr., Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0442USA. E-mail: rgogal@vt.edu Journal of Immunoassay & Immunochemistry, 27: 61–76, 2006 Copyright # Taylor & Francis LLC ISSN 1532-1819 print/1532-4230 online DOI: 10.1080/15321810500403755 61