Cell culture plates

Twenty-four-well cell culture plates with 8 μm inserts were purchased from EMD Millipore. For the migration assay, the HCCC and RBE cell lines (1 × 10⁵ cells per well) were added to the upper chamber in the presence of serum-free RPMI-1640 medium. A total of 500 μl complete medium (RPMI − 1640 + 10%FBS) was added to the lower chamber. The 24-well plates were placed in a cell incubator and incubated at 37°C and 5% CO₂ for 48 h. Next, cells in the upper compartment that failed to pass through the spoon were removed and washed with PBS 2-3 times. The cells that passed through the upper compartment and adhered to its lower surface were fixed with 4% tissue fixation solution (Elabscience Biotechnology Co., Ltd) for 0.5 h, stained with 0.1% crystal violet for 0.5 h, air dried at room temperature, and observed under the OLYMPUS BX43 (Olympus Corporation). Five fields of view were randomly selected for counting (magnification, ×100). The assay was carried out 3 times independently.

Caco-2 (HTB-37) cells were obtained from ATCC, and transporter knockout cells (C2BBe1 clone) were provided by SOLVO Biotechnology (Szeged, Hungary). Zosuquidar was obtained from Stratech (Ely, UK). Ritonavir, valspodar, Fumitremorgin C, novobiocin, Ko143, MK571, indinavir, E3S, CDCF and sulfasalazine were purchased from Sigma-Aldrich, St. Louis, USA. DMEM media, FBS, non-essential amino acids and Penicillin-Streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). HBSS was obtained from Thermo Fisher Scientific. Cell culture plates were purchased from Merck, Millipore. Acetonitrile and Methanol were obtained from Biosolve and ROMIL.