Label-free Measurement of Natural Killer Cell Cytotoxic Activity in Real-Time with the New xCELLigence System from Roche

Natural killer (NK) cells have been implicated in the recognition and annihilation of tumor cells. As demonstrated in a series of experiments, the real-time impedance-based, label-free xCELLigence System from Roche offers the only assay format so far to directly monitor NK cell mediated cytolysis without labelling the target cells and without using any chemical reporters. In addition, the technology that was invented by ACEA Biosciences, a cooperation partner of Roche in San Diego, USA, allows more accurate assessment of cytolytic activity by dynamically monitoring the entire history of cytolysis.

Natural Killer (NK) cells play a prominent role in various physiological and disease states. The assessment of their cytolytic activity is not only important for monitoring of immunocompetence in cancer, infectious diseases, and autoimmune diseases, but also in determining the proteins which mediate the cytolytic effect.

The standard methods used for measuring NK cell cytolytic activity are radioactive release assays, radio-labelling the target cells with for example 51Cr, and then adding the effector cells, such as NKs. The extent of NK activity towards target cells is assayed by the release of radioactivity by the target cells upon cytolysis. However, these methods are cumbersome not only due to the use of radioactivity but also due to high background because of the tendency of the label to permeate out of the target cells.

Monitoring of NK mediated cytotoxicity by measuring electronic impedance in real-time
As shown in a series of experiments, the use of the novel, real time, impedance-based xCELLigence System offers an alternative for label-free in vitro research of NK cell-mediated cytotoxic activity with significant contributions in ease of use and quality of data. The core of the xCELLigence System is the electrical impedance cell sensor arrays integrated into the bottom of microtiter plates which provide continuous, quantitative information about the biological status of attached cells. Any changes to cell number, size, morphology, or attachment quality will be detected in real-time without the use of reporter reagents. The integrated software allows data to be collected and saved automatically as often as every minute over virtually any time period.

Accurate assessment of NK-mediated cytolytic activity
The new impedance-based technology was used by researchers to dynamically and quantitatively monitor NK-mediated cytotoxic activity towards nine different target cell lines, including human cancer cell lines commonly used in the research field. Target cells were seeded in the wells of E-Plate microtiter plates and the impedance-based system dynamically monitored the cell growth every 60 minutes until the cells reached growth phase. The effector murine NK cells were then directly added to the wells at different E/T ratios, and the NK cell-mediated cytolysis was dynamically monitored. In addition, the technoloy was also successfully used in antibody mediated cell cytolysis assay (ADCC) for dynamic and quantitative monitoring of antibody mediated cancer cell killing activity.

In summary, these experiments demonstrate that the xCELLigence System can be used for assessment of NK cell-mediated cytolytic activity in both human and murine NK cell lines. The quantitative, dynamic measurement of NK cell-mediated cytolysis was achieved on the system without any labelling steps or reagents. Moreover, data obtained by the xCELLigence System clearly show that cytolytic activity can reach up to 70 %, which occurs after twelve hours of adding mNK cells. Such cytolytic activity which occurs after the standard incubation time of the assay can be easily missed by existing label-based endpoint assays. Therefore, the xCELLigence System not only offers label-free detection but, more importantly, also allows more accurate assessment of cytolytic activity by dynamically monitoring the entire history of cytolysis.

For more information on the technology, please visit the article webpage.