The measurement of complement-mediated lysis is dependent on several factors. Depending on the specific goals of the experiment, modifications to the protocol may be required. To test for a reduction in complement-mediated lysis, baseline lysis needs to be greater than 50% but not too high. To test for an increase in complement-mediated lysis, baseline lysis needs to be below 25% but not so low that it cannot be measured. More importantly, control cells not exposed to complement need to be included in the experiments to assess spontaneous lysis in the assay. Here, Creative Biolabs describes two methods to optimize lysis protocol for specific assays, including optimizing sensitization and decreasing complement regulation.
Optimizing Sensitization
In lysis experiments, if there is excessive complement activation, it can be solved by reducing the serum dose or the amount of sensitizing antibody used. Additionally, serum dilutions should be prepared in an appropriate buffer to reduce background lysis. When complement activation is insufficient, increased activation of the alternative pathway can be induced by adding zymosan. If the purpose of the experiment is to examine the effects of altered expression of endogenous complement inhibitors, glycolipid (GPI)-anchored proteins or cell lines lacking CD55 and CD59 can be used.
Decreasing Complement Regulation
To assess the effect of complement modulators, the function of other complement modulators needs to be blocked. For example, CD59 function can be blocked by incubating cells with saturating concentrations of blocking antibodies prior to the start of the experiment. A series of anti-CD59 antibodies have been developed to block CD59 function without activating complement. In addition, suitable antibodies can be used to block the function of DAF.
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