Today I am going to describe a flow cytometry-based NK cell cytotoxicity assay that we have developed on a BD Accuri™ C6 Plus cell analyzer. A number of cytotoxicity assays are commonly used to assess the ability of effector cells to recognize and kill target cells. Why a flow cytometry-based assay? Because flow cytometry offers the unique opportunity to simultaneously assess multiple parameters at the single cell level. This becomes critical when studying, for example, co-culture systems like the one we describe today. In this experiment, we have co-cultured NK cells together with the leukemic cell line K562 at different cell ratios for up to 4 hours. At the end of the co-culture, NK cell degranulation and K562 cell viability were simultaneously assessed in a single-tube assay. Before reviewing the data, let’s quickly review the staining protocol and instrument set-up.
Prior to co-culture, K562 cells were labeled with the PKH67 dye to facilitate separation of K562 cells from NK cells based on fluorescent signal. During co-culture, cells were stained with an anti-CD107a (BD Pharmingen™) antibody. CD107a will be used as a marker to assess NK cell degranulation and cytotoxic activity. After co-culture, cells were stained with BD Pharmingen™ APC Annexin V and BD Pharmingen™ 7-AAD to assess the viability of K562 cells.
Now let’s review instrument set-up. Before acquiring our samples, we run an automated, daily instrument QC. After adding one drop of QC beads to 350ul of PBS, we will load the tube and click on the instrument QC tab. This will open a QC window after which we can start QC by simply clicking on the "RUN" button. The system will automatically measure multiple parameters, such as noise, MFI and percent CV, to make sure the instrument is operating at its optimal performance standards. Within 2 minutes, the system has provided a report and confirmed that QC was passed; the system also allows us to track performance over time, which is very important when performing time course or longitudinal studies.
We can now load our sample and start acquiring data by clicking on the "RUN" button. Due to the fixed, pre-optimized settings, there is no need for manual instrument set-up or PMT voltage adjustment.
After acquisition, we can review our data. The broad dynamic range together with labeling of the K562 cells with PHK67 allows us to separate the two cell types based on size and fluorescence intensity. We can now gate on K562 and NK cells individually to simultaneously measure viability and degranulation, respectively. From the K562 cell gate, we can assess Annexin V and 7-AAD expression to identify live, early and late apoptotic cells. From the NK cell gate, the expression of CD107a can be measured as an indicator of cell degranulation and cytotoxic activity. As expected, at time 0, the majority of K562 cells are alive and NK cells express very low levels of CD107a. After 4 hours of co-culture, we observe a significant decrease in the percentage of live K562 cells, concurrent with an increase in CD107a expressing NK cells.
To conclude today’s video, I would like to highlight some of the BD Accuri™ C6 Plus system features that allowed us to develop this cytotoxicity assay. The BD Accuri™ C6 Plus is an easy-to-use personal flow cytometer equipped with a blue and a red laser and 4 fluorescence detectors. The fixed, pre-optimized detector settings and the automated instrument QC allow for a simplified workflow. Additionally, the ability to perform volumetric counting, enabled us in this experiment to directly quantify the amount of live target cells in different culture conditions and to identify the optimal effector-to-target cell ratio resulting in higher NK cell cytotoxic activity. Thank you for watching our video, I hope you find the information useful for the development of your assays.
For more information about the assay, please visit:
http://static.bdbiosciences.com/documents/BD-Accuri-C6Plus-NK-Cell-Cytotoxicity-DS.pdf
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