Ligandtracer green

The day before experiment, cells were seeded as 600 μL droplets with 10⁶ cells/mL near the edge of a circular cell dish (87 mm, Greiner, Germany) and were incubated at 37 °C overnight to allow their attachment to the dish surface. Two spots on the dish were used for SKBR3 cells, one was used for MDA-MB-231 cells and one spot was not treated (background reference area).
Prior to kinetic measurements, the cell medium was replaced with 3 ml fresh cell medium. The dish was placed in LigandTracer Green (Ridgeview Instruments)³⁶ . A baseline signal was collected for 30 min, and then a fluorescein-labeled conjugate was added in two increasing concentrations (9 and 27 nM) to record association. Dissociation of the conjugate was recorded after replacing the incubation solution with 3 ml fresh medium. Signals from cell and reference areas are recorded during every rotation, resulting in a background-substracted binding curve. Each concentration was incubated until sufficient curvature was obtained for subsequent extraction of kinetic parameters. Binding traces were analyzed with the evaluation software TraceDrawer 1.8.1 (http://www.ridgeview.eu/software/tracedrawer, Ridgeview Instruments) to determine k_a, k_d and K_D according to the « one-to-one » model or Langmuir binding model.

Human breast adenocarcinoma cells SK-BR-3 (ATCC HTB-30) and MDA-MB-231 (ATCC HTB-26)) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose (Sigma, St Louis, MO, USA). The medium was supplemented with 10% fetal bovine serum (Perbio, Brebieres, France), 2 mM L-Glutamine, 100 U/mL Penicillin and 100 µg/mL Streptomycin (Sigma). Cells were maintained in a 5% CO2 humidified atmosphere at 37 °C.
SK-BR-3 cells overexpress HER2 protein, and MDA-MB-231 cells are used as negative controls.
The day before the experiment on Ligand Tracer Green (Ridgeview Instruments), cells were seeded as 600 μL droplets with 8 × 105 cells/mL near the edge in 87 mm cell culture treated dishes (Greiner, Frickenhausen, Germany) and incubated at 37 °C overnight. Two droplets were prepared with SK-BR-3, one with MDA-MB-231, and one was left as a background reference (plastic).
Prior to kinetic measurements, the medium was carefully removed and 3 mL of fresh complete medium was added to the dish.

Real-time cell binding assay (RT-CBA) using LigandTracer^® Green (Ridgeview Instruments AB, Vänge, Sweden), and a blue (488 nm)-green (535 nm) detector was performed to evaluate the interactions of FAM-labeled 2c2s with PD-L1 protein expressed at the surface of aAPC/CHO-K1 cells. Cells were seeded in each target compartment of MultiDish 2 × 2 (1 × 10⁶ cells). Uncoated free areas of the Petri dish were measured to enable the subtraction of the background signal. The baseline measurement of cells in the absence of labeled aptamer was also performed. Kinetics of binding was analyzed with increasing concentrations (50, 200 or 650 nM) of the labeled aptamer. To evaluate the dissociation process, a fresh cell medium was provided. Affinity calculations (based on the association and dissociation rates) were performed using the TraceDrawer Software (Ridgeview Instruments AB, Uppsala, Sweden).

The interactions between FITC-labeled antibody and the− attached living bacteria cells were measured in a RT-CBA with LigandTracer® Green (Ridgeview Instruments AB, Vänge, Sweden), using a blue (488 nm)–green (535 nm) detector. The background signal of the fluorescent antibodies was corrected for through reference subtraction, using uncoated (E. coli measurements) or anti-His antibody-coated (S. carnosus measurements) bacteria-free areas of the Petri dishes as references. All LigandTracer measurements were conducted in PBS at RT and started with a short baseline measurement in the absence of a labeled antibody, to detect the background signal. The antibodies were added stepwise to obtain kinetic information at different concentrations which improves the accuracy of subsequent curve fitting (Onell and Andersson, 2005 ). In some of the measurements, the antibody solution was replaced with fresh PBS to assess the dissociation process.
A set of control experiments were performed to study the properties of Ab99. The anti-human epidermal growth factor receptor antibody cetuximab (purification from Erbitux, Apoteket AB, Solna, Sweden) was used as a negative control. Additionally, the binding of Alexa 488-labeled goat anti-rabbit IgG antibody (ab150077, Abcam) to adsorbed Ab99 was detected to study the adsorption properties.