Selection of laboratory assays for reliable assessment of complement-dependent cytotoxicity: impact of assay choice on CDC quantification

Complement-dependent cytotoxicity (CDC) results from cell lysis induced by the membrane attack complex (MAC), a pore-forming structure assembled at the terminal stage of the complement cascade that disrupts membrane integrity and causes osmotic cell death. Several methodological approaches are available to assess CDC efficacy in vitro , including (i) dye influx assays that report complement-mediated membrane permeabilization (e.g., propidium iodide staining), (ii) dye-release assays using preloaded fluorescent probes (e.g., calcein-AM), and (iii) assays based on cellular metabolic activity (e.g., MTT, XTT, or Alamar Blue) or ATP content (e.g., CellTiter-Glo). Because complement acts rapidly, often killing target cells within seconds to minutes, and because some staining procedures are cytotoxic or require time-consuming steps that may introduce artefacts, selecting an appropriate, high-throughput assay is not trivial. In this study, we evaluated representative methods from each assay class (dye influx, dye release, and metabolic readouts) to measure CDC triggered by the therapeutic anti-CD20 antibody rituximab in two human lymphoma cell lines expressing the CD20 antigen. Based on our findings, we discuss the strengths and limitations of each approach, with particular emphasis on their susceptibility to false-positive and false-negative results.