Cell-mediated cytotoxicity: A highly sensitive and informative flow cytometric assay

摘要: Determination of target cell lysis by cytolytic effectors has typically been achieved two methods: the release various markers from cell, as in 51chromium assays and uptake into trypan blue single cell/conjugate binding assays. Problems associated with these might include: (1) poor uptake, (2) nonspecific release, (3) statistics, (4) length assays, or (5) subjectivity. These difficulties prompted development a new sensitive flow cytometric assay employing fluorochromes. PKH-1, fluorochrome which fluoresces green, binds to cytoplasmic membrane does not leak transfer, is used identify population. Propidium iodide red detect non-viable cells. Use fluorochromes parameter analysis allows for identification four subpopulations sample: live effectors, dead targets targets. By enumeration following information can be calculated: percent lysis, effector-to-target ratios, viability effector cells at termination assay, viable ratios. The results show that PKH-1 labeling had no effect on effector-target interactions. Excellent correlation was found between this method chromium however, due earlier detection lytic event, provides distinct time advantage over current methods.