Hyclone fetal calf serum

Human embryonic kidney (HEK) 293 cells (ATCC, Teddington, UK) were maintained on 90 mm tissue culture plates at 37 °C and 7% CO₂ in a humidified atmosphere. They were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mix F12 (DMEM: 1:1) with GlutaMAX I (Thermo Fischer Scientific) containing 10% HyClone fetal calf serum (GE Healthcare, Hatfield, UK). For FlexStation (Molecular Devices, Wokingham, UK) studies, cells were transfected using polyethylenimine (PEI; Merck, Gillingham, UK): 30 μL PEI (1 mg/mL), 5 μL cDNA (1 mg/mL; subcloned into pcDNA3.1), and 1 mL DMEM were incubated for 10 min at room temperature, added dropwise to a 70–90% confluent plate, and incubated for 2 days. Cells were then transferred to poly-l-lysine (Cultrex)-coated 96-well plates (Greiner BioOne, Stonehouse, UK) and allowed to adhere overnight before use.

CB[6] and CB[7] were synthesized at the Nikolaev Institute of Inorganic Chemistry SB RAS (Novosibirsk, Russia) through the standard procedure described in [25 (link)]. The structures of CB[6] and CB[7] were verified with ¹H NMR in D₂O at 25 °C (Figure S2 in Supplementary) using a 500 MHz Bruker Avance III spectrometer. Medium, phosphate-buffered saline, and L-glutamine were obtained from Biolot (Saint Petersburg, Russia). HyClone fetal calf serum was obtained from GE Healthcare (Chicago, IL, USA). WST-1 reagent was purchased from Takara Bio Inc. (Kusatsu, Japan). Anti-CD3 antibody was obtained from MedBioSpectr (Moskow, Russia). Recombinant IL-2 was obtained from Biotech (Saint Petersburg, Russia).