Ž . Journal of Immunological Methods 209 1997 137–154 Determination of parameters that characterize effector–target conjugation of human NK and LAK cells by flow cytometry Gonzalo Rubio a , Josefa Garcia-Garcia a , Jesus Galvez b , Nieves Lorenzo a , Francisco Lajarin a , Pilar Garcia-Penarrubia a, ) ˜ a Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, 30100 Espinardo, Murcia, Spain b Laboratory of Physical Chemistry, Faculty of Science, 30100 Espinardo, Murcia, Spain Received 4 February 1997; revised 13 June 1997; accepted 1 September 1997 Abstract Effector–target conjugation between different cell populations of human NK cells and K562 tumor cells has been studied Ž . Ž . from binding isotherms obtained from data of effector a and target b conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios. Non-linear and linear regression methods were applied to these isotherms to calculate the binding parameters that characterize the process of conjugation, namely, the maximum effector and target conjugate frequencies, the dissociation constant of the conjugates formed, the binding units and the area under the Ž. binding isotherms. The results obtained show that: 1 flow cytometry analysis of effector–target conjugation is faster, unbiased and more suitable than microscopic counting of conjugates, thereby permitting the analysis of larger number of Ž. conjugates in shorter times, 2 the binding parameters derived from conjugate frequencies obtained by flow cytometry Ž. analysis differ from those obtained by microscopy, 3 the discrepancies between the two methods are due to the presence of Ž. several cells engaged in multicellular conjugates that are detected as single particles by flow cytometry and 4 the analysis of population distributions of the conjugates formed at different values of the effector-to-target ratio permit the above discrepancies to be corrected. q 1997 Elsevier Science B.V. Keywords: Effectorrtarget conjugation; Binding parameters; NK cell conjugation; NKL conjugation 1. Introduction Cell to cell interactions are of crucial importance for a number of leukocyte functions. Effector cell recognition of and binding to target cells leading to the formation of conjugates is the first step of antigen Ž .Ž . recognition and cell mediated cytotoxicity CMC Berke, 1980, 1994; Trinchieri, 1989 . Hence, quantitation of the conjugation process that permits us to characterize and measure physical interactions between effector and Ž target cell populations is fundamental to CMC studies Berke, 1980, 1994; Bonavida et al., 1983; Macken and . Perelson, 1985 . ) Corresponding author. Tel.: q34-68-363960; fax: q34-68-364150: e-mail: pigarcia@fcu.um.es 0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0022-1759 97 00153-1