Journal of Immunological Methods, 22 (1978) 63--71 63 © Elsevier/North-Holland Biomedical Press COMPARING STIMULATION OF LYMPHOCYTES IN DIFFERENT SAMPLES: SEPARATE EFFECTS OF NUMBERS OF RESPONDING CELLS AND THEIR CAPACITY TO RESPOND STELLA C. KNIGHT and JOHN FARRANT Clinical Research Centre, Watfbrd Road, Harrow HA1 3UJ, Middlesex, U.K. (Received 11 November 1977, accepted 1 February 1978) A range of concentrations in culture of human lymphocytes from the peripheral blood has been used to provide information on two major factors contributing to the total response to stimulation with mitogens; these factors are the numbers of responding cells, and the efficiency of these responding cells. The marked effect of cell concentration in culture on the thymidine incorporation of stimulated lymphocytes ranged from a background response to a maximal response mainly over 1 log unit of cell concentration (cells/ml). At higher cell concentrations the measured response fell. Since the absolute number of lymphocytes that respond to stimulation was not known, the relative positions of these responses (incorporations of thymidine) on the axis of concentration of cells were used to estimate the relative numbers of responding cells in different samples. The efficiency of responding cells in different samples was compared using the shape of the response curve with changing concentration of cells. With this approach, frozen and thawed samples of lymphocytes were compared with non-frozen cells and the results indicated a loss in the numbers of responding cells after freezing and thawing, but no change in their efficiency of incorporating thymidine on stimulation with phytohaemagglutinin or allogeneic cells. INTRODUCTION The recovery of responsiveness to mitogenic stimuli of lymphocytes in a sample of cells after freezing and thawing is usually measured at a single con- centration of cells in culture by comparing the incorporation of thymidine into DNA on stimulation before and after freezing (for review, see Wether et al., 1973). One problem with this approach is to decide whether a changed response in this assay is due to a relative increase or decrease in the numbers of responding cells, or to an altered efficiency of the cells. The number of cells responding to stimulation cannot be estimated either from total cell counts or dye exclusion. Also, changes in the numbers of responding cells cannot be determined using a single concentration of cells in culture, since uptake of thymidine varies with concentration of responding cells in a non- uniform way. We have therefore compared the samples of cells by mea- suring the responses to stimulation over a large range of cell concentrations in culture, and used the patterns of responsiveness over the whole range