FlowX Human NK Cell Killing Flow Cytometry Kit

Figure 1a. Example of Killing assay data using NK cells and K562 cells Human NK cells were isolated from whole blood using a Ficoll gradient. To enrich for NK cells, both monocytes and T cells were depleted from the PBMCs using a 2-step purification. Specifically, MAGH105 was used to deplete CD14+ cells, and the remaining CD14- cells were counted, and then depleted of CD3+ T cells using MAGH101. CD14-CD3- NK cells were activated for 14 days in a 6-well plate which was coated with 10 µg/mL anti-NKp46 (MAB1850) in CCM032 media (R&D Systems) supplemented with hIL-2 (27 ng/mL; 202-GMP; R&D Systems), hIL-12 (10 ng/mL; 219-GMP; R&D Systems), hIL-18 (10 ng/mL; 9124-IL; R&D Systems), and hIL-21 (10 ng/mL; 8879-GMP; R&D Systems). At day 14, NK cells were labeled with Janelia Fluor NK cell dye and K562 target cells were labeled with Mito Mark target cell dye as described above. NK and K562 cells were then co-incubated for 2 hours at Effector (NK) to Target (K562) ratios of 1:0, 0:1, 0.5:1, and 5:1 (1 = 0.25 x 106 cells). LAMP-1/CD107a PE or Mouse IgG2b PE were included in the cell culture media for the entirety of the killing assay for detection of LAMP-1/CD107a proteins, or a negative control, respectively. The cells were harvested, stained with Viability Staining Dye, and surface stained with Human NCAM-1/CD56 Alexa Fluor® 700 and Human CD3 epsilon Alexa Fluor® 405. The stained cells were then fixed with 1X Flow Cytometry Fixation Buffer. Cell Count Particles were added, and cells were then run on the BD LSRFortessa™. A. Gating strategy with Cell Count Particles. Rectangular gate is on the Cell Count Particles; lower left polygon gate is based on the NK cells; far right polygon is on K562 cells.

Figure 1b. Example of Killing assay data using NK cells and K562 cells. NK cells labeled with the Janelia Fluor NK cell dye. The FSC/SSC dot plot shows the NK cell population and histogram shows NK cells labeled with the Janelia Fluor NK cell dye.

Figure 1c. Example of Killing assay data using NK cells and K562 cells. K562 cells labeled with Mito Mark target cell dye. The FSC/SSC dot plot shows the K562 cell population and histogram shows K562 cells labeled with Mito Mark target cell dye.

Figure 1d. Example of Killing assay data using NK cells and K562 cells. LAMP-1/CD107a is upregulated on activated NK cells at an E:T ratio of 5:1. The purity of the activated NK cells is shown in a dot plot using Human CD3 epsilon Alexa Fluor® 405 vs. Human NCAM-1/CD56 Alexa Fluor® 700. The level of LAMP-1 expression in CD56⁺ NK cells is shown in the far-right dot plot.

Figure 1e. Example of Killing assay data using NK cells and K562 cells. Graphical representation of killing by NK cell effectors. The graph depicts K562 baseline staining, K562 baseline death, K562 cells that are live in the killing assay, and K562 cells that are dead due to NK killing. This is calculated using the 0:1 ratio and 5:1 ratio of E:T cells (see histograms).

Figure2a. Killing efficiency of CAR-NK Cells Human NK cells were enriched from PBMCs and transduced with CD19 CAR (CAR-NK). (A) CAR-NK expression was determined using rhCD19-Atto 647N (Cat # ATM9269) and hCD56 PE Monoclonal Antibody (Cat # FAB24086P).

Figure2b. Killing efficiency of CAR-NK Cells CAR-NKs were labeled with Janelia Fluor, Daudi target cells were labeled with Mito Mark Green, and cells were co-incubated for 2 hours at Effector to Target ratios shown above along with anti-Human LAMP-1 PE Antibody (Cat # IC4800P). Cells were harvested and stained with hCD56 and hCD3.