Characterization of aerolysin resistant T-cell clones. (A) Flow cytometric analysis of pooled aerolysin-resistant T-cell clones. Day-16 colonies were washed and stained with FLAER. — represents expanded T-cell clones grown without aerolysin; –, expanded T-cell clones grown in the presence of aerolysin. (B) Mutation from CFU-GM/AE 9/7 lost MslI restriction site, which is CACAA′GGATG. Thus, MslI was used to screen aerolysin-enriched T-cell clones grown from the same donor. A total of 8 aerolysin-resistant T-cell clones were individually plucked and genomic DNA was extracted. A representative example is shown. Exon 4 of the PIG-A gene was amplified with the proofreading polymerase Pfu Ultra using the primers shown in Figure 1 and Table 1. The products were purified and digested with MslI. The PCR product from CFU-GM/AE 9/7 (lane P) which serves as a positive control were mixed in a 1:1 ratio with the normal allele, because the PIG-A gene is on the X chromosome and the X chromosome is randomly inactived in females. The TF1 cell line (lane C) serves as a normal control. Lane U represents uncut DNA from the T-cell clone and lane T shows the consequence of the Ms1I digest of DNA from a representative T-cell clone. M indicates molecular size markers.