15] Use of dyes and radioisotopic markers in cytotoxicity tests

Abstract

In this chapter, the use of trypan blue dye exclusion; release of 51 Cr, 125 IUdR, and CH 3 [ 3 H]Thd; and uptake of CH 3 [ 3 H]Thd as indicators of humoral and cell-mediated immunological cytotoxicity is described. The radioisotopic markers that label cytoplasmic proteins ( 51 Cr) or nuclear material ( 125 IUdR or CH 3 [ 3 H]Thd) are readily employed in cell-mediated cytotoxicity assays and are generally accepted as good indicators of cell death. For example, 51 Cr has the advantage of being able to label both nonreplicating and replicating cells with little toxicity. However, it displays early and high spontaneous release with most nonlymphoid cells, making it unacceptable for long-term assays over 6–8 hr. In addition, it cannot be used at low target cell concentrations. 125 IUdR is incorporated exclusively into cellular DNA and released only with cell death and lysis. As the reutilization of the isotope is negligible, the amount released into the supernatant during incubation is an accurate reflection of cell death. Similar properties have been established for CH 3 [ 3 H]Thd. However, both these labels may be toxic, may inhibit replication, or may induce autoradiolysis if used at too high a concentration. Nevertheless, they can be used essentially to quantify nuclear disintegration, a valid, reliable, and well-accepted measure of cell death.