A novel assay to quantify cell death after transient expression of apoptotic genes in B- and T-lymphocytes.

摘要

We developed an assay allowing the detection and quantification of cell death after transient expression of apoptotic genes in B- and T-lymphocytes. For efficient gene transfer, B- and T-cells were electroporated under optimized conditions. To blind out the high background of non-transfected cells and cell death caused by the electroporation procedure itself, the green fluorescent protein (GFP) was co-transfected with the gene of interest. However, if the gene of interest was a potent apoptosis inducer, most successfully transfected cells were killed before GFP was expressed to levels sufficient for standard flow cytometry analysis or apoptosis assays. After staining of the transfected cells with propidium iodide (PI), very few GFP+/PI+ cells were detectable. To overcome this problem, the cell death rate induced by the transiently expressed gene was determined as the reduction of living green cells in the apoptotic versus a reference sample. This was achieved by an advanced flow cytometrical analysis quantifying the number of surviving green cells in normalised sample volumes directly relating to the number of initially transfected cells. Functioning of the assay was demonstrated by transient transfection of the potent apoptosis inducers TNF-receptor-associated death domain protein (TRADD) and a fusion protein of the transmembrane domain of the latent membrane protein 1 (LMP1) of Epstein-Barr virus and the signaling domain of TNF-receptor 1. We successfully applied the assay to the Burkitt lymphoma cell line BJAB and the T-leukemia cell line Jurkat.