Measurement of Complement Lysis of Nucleated Cells

Lysis is the final end point of complement attack, indicating that the mechanisms acting to protect the cell have been overwhelmed. There is, of course, enormous biological significance in the deposition of complement fragments on cells in vivo because these serve as opsonins and opsonization is accompanied by the generation of anaphylatoxins and chemotaxins, often without lysing the cell. Measurement of the generation of anaphylatoxins, cell-bound complement fragments and hemolysis of erythrocytes will be described in other chapters. This chapter will focus on methods for measuring the complement-mediated lysis of nucleated cells in vitro. Nonnucleated cells, such as erythrocytes, lyse with relative ease when exposed to sensitizing antibody and complement. In the case of erythrocytes, this causes release of hemoglobin, which can easily be measured in the supernatant (Chapter 4). In comparison, measuring the lysis of living nucleated cells is more challenging. Nucleated cells, unlike erythrocytes, do not contain a natural chromophore. Nucleated cells have active budding processes to shed membrane attack complexes (MAC) from the membrane and they have ion-pumps that actively reclaim released ions, stabilizing the osmotic balance and preventing rupture of the plasma membrane.