Luciferase-based cytotoxicity assay

The following protocol provides a simple procedure for the evaluation of the functionality of any type of effector cell recruiting antibody constructs for targeted cell killing. 

As an target cell, any adherent or suspension cells expressing the target antigen of interest and which can be engineered to overexpress firefly luciferase (EC 1.13.12.7) can be used. As an effector cell, any type of cell with inducible lytic activity can be employed. We routinely use PBMCs and purified cytotoxic T cells to evaluate CD3-engaging antibody derivatives against a number of different tumour-associated antigens (TAA).

For cell lysis experiments, seed 10 000 to 50 000 cells co-expressing the target antigen and firefly luciferase per well of a sterile 96-well plate (flat white Costar, Corning, USA) in 0.05 ml growth-supporting cell culture medium (e.g. RPMI1640 supplemented with antibiotics and serum). The number of cells seeded depends on the duration of the experiment and the proliferation rate of the target cells. Before starting an experiment, determine the optimum seeding density. The number of cells should be chosen such that overgrowth and excessive consumption of the culture medium will be avoided. When using adherent target cells, pre-incubate for 1 to 2 hours at room temperature after seeding to minimise the edge effect (Ref: Lundholt BK, Scudder KM, Pagliaro L. A simple technique for reducing edge effect in cell-based assays. J Biomol Screen. 2003;8(5):566-570). When using suspension target cells, incubation at room temperature immediately after seeding is not necessary. The cells are then incubated at 37°C, 5% CO2 and saturated realtive humidity for 18-22 h. The next day, effector cells and antibody constructs are added to the target cells without removing the previous medium. Effector cells are resuspended in the same cell culture medium used for the target cells and added at an effector to target cell ratio (E:T) of 1:1-10:1. A volume of 0.05 mL of effector cell suspension is applied per well. Antibody constructs are diluted in 0.02 mL (per well) of cell culture medium and also added to the plate. Mix the plate by gentle double orbital shaking and incubate for a further one to two days at 37°C, 5% CO2 and saturated realtive humidity. The intracellular luciferase activity is then measured to quantify the lysis rate of the target cells. To do this, D-luciferin (Biosynth, USA) is added to each well to a final concentration of 0.5 mM and the plate is incubated for 20-30 minutes before the luciferase-induced light emission is measured. We use the Spark Multimode Microplate Reader (Tecan, Switzerland) to detect and quantify the luminescence.

Calculation of cell viability

RLI_sample x 100%

-------------------------- = Viability [%]

RLI_control

Calculation of specific lysis

100 – Viability [%] = % specific lysis

Legend

RLI : relative luminescence intensity

Sample : Effector cells + Target cells + Antibody

Control : Effector cells + Target cells