UNIT 7.10 Measurement of Proliferative Responses of Cultured Lymphocytes Linda Mesler Muul, 1 Christopher Silvin, 1 Stephen P. James, 2 and Fabio Candotti 1 1 GMBB, National Human Genome Institute, Bethesda, Maryland 2 University of Maryland School of Medicine, Baltimore, Maryland ABSTRACT Measurement of proliferative responses of human lymphocytes is a fundamental tech- nique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to measure the proliferative response of lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying support protocol which contains methods for pulsing cultures with [ 3 H]thymidine and determining incorporation of [ 3 H]thymidine into DNA or assessing cell proliferation by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT). The protocols described here provide an estimate of DNA syn- thesis and cell proliferation in an entire cell population, but do not provide information on the proliferation of individual cells. A protocol for CFSE labeling allows specific sub- populations of cells to be separated viably for further analysis. Curr. Protoc. Immunol. 82:7.10.1-7.10.24. C  2008 by John Wiley & Sons, Inc. Keywords: lymphocytes  proliferation  mitogens/antigens  CFSE  mixed lymphocyte reaction (MLR) INTRODUCTION Measurement of proliferative responses of human lymphocytes is a fundamental tech- nique for the assessment of their biological reaction to various stimuli. Most simply, this could be accomplished by measuring the number of cells present in a culture before and after the addition of a mitogen or antigen. However, this can be both laborious and inac- curate, inasmuch as proliferating cells may constitute only a small component of the total cell population, and there may also be cell death. In practice, therefore, cell prolifera- tion has most commonly been determined by estimating incorporation of [ 3 H]thymidine into DNA, a process which is closely related to underlying changes in cell number. With increasing concerns about radiation use and disposal, alternative nonradioactive, high-throughput protocols have been developed. These assays are colorimetric protocols that primarily use reduction of tetrazolium salts to formazans as a surrogate measure of cell proliferation. Both [ 3 H]thymidine and the tetrazolium assays provide an indirect estimate of DNA synthesis and cell proliferation in an entire cell population, but they do not provide information on the proliferation of individual cells. In cases where one needs to determine which subpopulation of cells is proliferating, the intracellular dye CFSE (carboxyfluorescein diacetate, succimidyl ester) offers direct measurement of cell division and may be used with combinations of various cell surface markers. This unit contains several different prototype protocols for measuring the proliferative re- sponse of populations of lymphocytes following exposure to mitogens (Basic Protocol 1), allogeneic (Alternate Protocol 1) or autologous (Alternate Protocol 2) cells, and antigens or soluble factors (Alternate Protocols 3 and 4). Each of these protocols can be used in Current Protocols in Immunology 7.10.1-7.10.24, August 2008 Published online August 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142735.im0710s82 Copyright C  2008 John Wiley & Sons, Inc. Immunologic Studies in Humans 7.10.1 Supplement 82