The polymerase chain reaction applied to identification of specific alleles of the bovine milk protein genes.

Perforin was purified to homogeneity from the murine cytotoxic T-lymphocyte cell line, CTLL-2 B 1 /6, by minor modifications of published methods [3]. In order to identify the maximum dose of perforin causing no lysis of KS62 cells (non-lethal dose), cells were incubated in Krebs-Hepes buffer with various doses o f perforin for 1 0 min at 37°C. Cell lysis was estimated by Trypan Blue uptake. Similarly, to find a non-lethal dose in complement-mediated attack, the cells were optimally sensitized with a rabbit polyclonal antiserum raised to KS62 cells ( 4 T , 30 min) followed by formation of CSh-7 sites on the membranes by incubation with normal human serum depleted o f C8 and CY (3 min, 37°C). Aftcr washing away the serum, C8 was added back in excess followed by limiting amounts o f CY. Permcabilization of the cells was followed by flow cytomctry (FACS 440; Becton Dickenson). PI was added to cells at 4°C at time zero together with perforin o r CX/CS. Fluorescence-activated cell sorting (FACS) analysis of KS62 cells incubated with non-lethal amounts of perforin in the presence of PI demonstrated that more than 90% became pcrmeablc t o the dye. Increased cell fluorescence was detcctable within 2.5 inin o f pcrforin addition and reached a maximum by 6-7 min. remaining at this level for the duration of the experiment (Fig. I a). MAC pcrmeabilization was similarly examined in KS62 cells bearing CSb-7 sites by incubation with CX and C9 in the presence of PI. Increased fluorescence was detectablc in about 1 0 % of cells within 3-4 min of C8/CY addition and the number o f PI-positive cells increased slowly to a plateau at which 40'% o f thc cells were positive by 25 min. The PIpositive cells remained viable (assessed by Trypan Blue exclusion) for at least 24 h after perforin or MAC attack. Estimates of the life-time of the functional channel formed by non-lethal amounts o f perforin in KS62 cells were performed by incubating cclls with perforin at 37°C. adding PI t o aliquots of cells at intcrvals and measuring PI uptake by FACS. T h e number o f cells remaining PI-permeable started to decrease between the first and second minute after perforin addition and when PI was added 3 min after initiating perforin attack. uptakc was minimal. demonstrating that the channel was transient with a half-life o f about YO s (Fig. 16). The life-time of the MAC functional channel was also examined by FACS. T h e percentage of cells taking up PI was highest when the dye was added 3-4 min after addition of C8 and CY to KS62 cells bearing CSb-7 sites (25% positive). Addition of PI at later time points revealed a slow decrease in the percentage of fluorescent cells. However, even 80 min after initiating attack 1014% of the cells were permeable to added dye. These results demonstrate that both non-lethal perforin and complement attack can cause transient membrane channels on KS62 cells. T h e kinetics of channel formation and removal were, however, very different. Perforin attack rendered > 90% of cells permeable to PI, but the channel was very short-lived (half-life YO s). Non-lethal amounts o f MAC rendered far fewer cells PI-permeable, permeabilization was slower and the channels persisted for longer. T h e reasons for the differences in the kinetics of cell recovery from non-lethal attack by these two pore-forming agents remain to be established.