Black 96 well cell culture microplate

An EVOM Voltohmmeter (World Precision Instruments), equipped with STX2 chopstick electrodes (World Precision Instruments) was used to measure the transepithelial resistance (TER). Briefly, 5 × 10⁴ cells were plated into a 0.4-μm pore size insert (Greiner Bio-One Ltd) and cultured to 100% confluence. Electrodes were placed in the upper and lower chambers and resistance was subsequently measured using a Volt-Ohm meter. Inserts without cells in medium were set as a blank control. Following the analysis of TER, the medium in the upper chambers was replaced with normal medium containing 0.2 mg/ml fluorescein isothiocyanate (FITC)-dextran 10 kDa. Then, 50 µl medium from outside the insert was transferred into a black 96-well cell culture microplate (Greiner Bio-One) in duplicate every 2 h for 10 h. The basolateral dextran passage was analysed using a GloMax®-Multi Microplate Multimode reader (Promega Corporation), with an excitation wavelength of 490 nm and an emission wavelength of 510–570 nm. Each measurement was normalized to the 0 h via subtraction.

The effector function assay was performed as described previously [41 (link)]. To perform the CDC assay, the cells were seeded in a black 96‐well cell culture microplate (Greiner Bio‐One, Alphen aan den Rijn, Netherlands) and incubated with NN2101. The cells were then treated with 20% (v/v) human serum complement (Quidel, San Diego, CA, USA) and incubated in a humidified CO₂ incubator. Next, the cells were stained with Calcein AM (Invitrogen), propidium iodide (Sigma Aldrich, St. Louis, MO, USA), and Hoechst 33342 (Invitrogen). The stained cells were counted using a Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, KS, USA).
The ADCC assay was performed using the ADCC reporter bioassay, core kit (Promega, Madison, WI, USA). The cells were seeded in a 96‐well Cell Culture Microplate White (Greiner Bio‐One) and treated with NN2101 at RT. Jurkat cells stably expressing the FcγRIIIa receptor (V158 variant) and an NFAT response element driving the expression of firefly luciferase were used as effector cells (Promega). The Jurkat cells (6 × 10⁴ cells/well) were co‐cultured with the target cells (effector/target ratio = 6 : 1) for 6 h in a humidified CO₂ incubator. Activation of effector cells was determined by quantifying the luciferase activity using GloMax reagent (Promega), following the manufacturer's instructions.