A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

“…The measurement of NK cytotoxic activity using this EGFP-flow cytometric assay not only showed a high correlation with the standard 51 Cr release assay, but also enabled to cut the time period required for assay, as previously reported (13). Several publications have highlighted a significant correlation between 51 Cr release assays, fluorescent dye release assay, and flow cytometric assay (13,15,17,19,24). Our development and optimization of the test demonstrate several interesting and crucial features of these analytical conditions: (i) A reduced volume of blood (i.e., 4 ml), for 3-6 assays depending on the E/T ratio 5:1 or 10:1 used and depending on the percentage of NK cells in the blood of each volunteer (4-37%).…”

Cell‐mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere‐adjusted counts on three‐color flow cytometry

“…Percentage-based cytotoxicity reached peak levels at the end of 2-4 h of coincubation and started declining after 4 h, depending on the target cell population (5,8,15). In comparison studies with CRA, samples were coincubated for 4 h, whereas in some experiments samples were coincubated for 2 h (Fig.…”

“…However, some investigators expressed concern about such dyes leaking and labeling other cells in the environment during coincubation (8). In a limited number of experiments in which we utilized such dyes, we also faced the same difficulties.…”

“…This assay can also be used to study the formation of conjugates between effectors and targets (8). Since cellto-cell interaction is required for target-cell kill in the cellular immune reaction, conjugate formation is a necessary step in this process.…”

“…Several different methods have been suggested to identify target and effector populations, such as the use of differences in light scattering properties. Recently, Zamai et al (8) developed a flow cytometric assay using preculture, direct staining, with a CD56-antibody and light scatter changes for the detection of cytotoxicity against K-562 cells, which can be useful, although not easily adapted to other systems (8,9). In later studies, researchers utilized mAb staining in combination with either PI or annexinV-FITC (AnnV) (2,8).…”