Subtraction of autofluorescent dead cells from the lymphocyte flow cytometric binding assay

Flow cytometry allows the quantitative analysis of lymphocyte-target cell conjugates and the identification of the lymphocyte subset involved in the binding phenomenon. We recently described a methodology to identify the effector cells bound to K562 targets based on target cell autofluorescence coupled with lymphocyte staining by means of fluorescent monoclonal antibodies. Here we describe an implementation of the methodology that allows the subtraction of spontaneously dead targets to which lymphocytes may or may not adhere, thereby preventing the overestimation of the binding phenomenon and limiting its evaluation to living effector-target conjugates, thus preserving the specificity of the phenomenon.