Multiploidy of the cells was examined as reported previously (Kanazawa et al., 2000 (link)). Briefly, cells treated with psychosine for 2 d were fixed in 70% ethanol overnight. After washing with PBS, the cells were incubated with RNase A (DNase-free; Nacalai Tesque, Kyoto, Japan) to remove RNA. The resultant cellular DNA was stained with propidium iodide (Nacalai Tesque) for subsequent flow cytometry (FCM) analyses using a FACScan or a FACSCalibur flow cytometer for FL-2 channel detection. To exclude doublets, cells were gated using the FL-2 Area and FL-2 Width. Average cell ploidy was calculated from the abundance ratio of each nuclear phase. In the multiploidization experiments, the comparison was made only using control cells cultured side by side. The PPIN value was developed to profile psychosine-induced multiploidy in six different B-cell lines, in which normalization of the cell-to-cell differences of psychosine sensitivity seemed appropriate. The concentration of psychosine to achieve maximum multiploidy was determined. Values of the maximum percentage of each cell with a ploidy >4N were divided by the concentration of psychosine to calculate the PPIN for normalization of cell variations in psychosine sensitivity and degree of multiploidy. When PPIN was not needed, average ploidy was calculated and is depicted on the upper right of the histograms.