Antibody-dependent cell cytotoxicity (ADCC) assays are frequently used in immunological studies, oncology research, and the development of therapeutic antibodies. The use of cryopreserved effector cells in ADCC assays can provide a convenient, ready-to-use alternative to freshly isolated or cultured effector cells. We have optimized a protocol for ADCC assays using cryopreserved NK effector cells from BioIVT. The optimized protocol with recommended adaptations is provided below. For data and information on the performance of the assay, please refer to our technical article “ADCC assay using cryopreserved NK cells” .
Before you start, there are several key points to be considered:
Target cell preparation (Timing: 2 h):
Figure 1.Sample plate map including target cell numbers, effector-to-target cell (E:T) ratios, and anti-EGFR concentrations.
Figure 2.Distribution of A549 cells plated in 96-well plate and incubated at room temperature for 1 hour. The picture was taken from the edge (A) and middle (B) part of the well using a Millicell® DCI Digital Cell Imager. An even distribution of cells should be observed throughout the well, without any aggregation of cells (particularly at the edges of the well).
NK cell preparation (Timing: 1 h):
Killing reaction (Timing: 2 h):
Measure luminescence (Timing: 2 h):
After 24 hours, target cell death should be observable using a microscope (Figure 3)
Figure 3.NK killing of A549 target cells, mediated by anti-EGFR, after 24 hours incubation. The E:T ratio was 18:1. The concentration of anti-EGFR antibody was 1 µg/mL (A) using a 0 µg/mL anti-EGFR antibody control (B). Clear killing of target tumor cells was observed in the presence of antibody as seen by cellular detachment and clamping of dead cells and debris. In contrast, target tumor cells appear healthy with a complete monolayer in absence of anti-EGFR antibody. Images were taken using a Millicell® DCI Digital Cell Imager.
Data can be plotted three ways:
Live cell numbers can be interpolated using luminescence signal from a standard curve of A549 cells expressing the luciferase reporter gene.
Figure 4.Representative data for ADCC assay with serial titration of anti-EGFR antibody. The NK cells isolated from three donors (A, B and C) were incubated with luciferase-expressing A549 target cells with increasing concentrations of anti-EGFR antibody for 24 hours. For all experiments, the E:T was 18:1. Luciferase activity in surviving target cells was determined by luminescence measurement. The raw luminescence data were fitted to a sigmoidal three-parameter curve. EC50 values were obtained using following formular: Y=Bottom + X*(Top-Bottom)/(EC50 + X) (D). ! Caution: Both ends of the curve should be flat, indicating a reaction close to minimum / maximum response. This is critical for deriving an accurate EC50 for the reaction. If the slope is still steep, consider repeating the experiment using a lower or lower antibody concentration as appropriate.
Cytotoxicity % = [(Max-BG) - (Expt-BG)] / (Max-BG) * 100
where Max represents the average signal from target cells only, Background (BG) represents the average signal from wells with
no target cells, and Expt denotes experimental samples.
Figure 5.Cytotoxicity % for each sample were fitted to a sigmoidal three-parameter curve. EC50 values were obtained using following formular: Y=Bottom + X*(Top-Bottom)/(EC50 + X) (D). EC50 values match those obtained from plotting raw luminescence signal vs. antibody concentration (Figure 4D).
ADCC % = Cytotoxicity % (with antibody) – Avg (Cytotoxicity % (without antibody))
Figure 6.ADCC % for each sample were fitted to a sigmoidal three-parameter curve. EC50 values were obtained using following formular: Y=Bottom + X*(Top-Bottom)/(EC50 + X) (D). EC50 values match those obtained from plotting raw luminescence signal vs. antibody concentration (Figure 4D).