Analysis using the spreadsheet method of the effects of PMA treatment on hyaluronan binding to CD44 BW5147 cells were left untreated or treated with 160 nM PMA for 1 h or 48 h and then stained for three-colour flow cytometry as described in the Methods. For the negative control, untreated cells were stained with FITC-BSA followed by PE-streptavidin. The flow cytometry data is displayed as follows: A, histogram plots showing CD44 expression or FITC-hyaluronan binding for each treatment. B, two-parameter dot plots correlating FITC-hyaluronan binding against CD44 expression. C, the data was analysed using the spreadsheet method (see text for further details). The values given are arbitrary fluorescence units. Where there were 3 cells or fewer for a given PE fluorescence channel, the point is plotted at 0 on the y-axis.