Erythrosin b

Fetal membranes were washed with ice-cold phosphate-buffered saline (PBS, Corning, NY, USA) with 1% penicillin-streptomycin solution (10,000 U/mL penicillin, 10,000 U/mL streptomycin, Corning, Steuben County, NY, USA). Amniotic membrane was mechanically peeled off the underlying chorion layer; to remove any blood clots, tissues were incubated for 10 min at room temperature with PBS/ ethylene diaminetetraacetic acid (EDTA) 0.5 mM. The amniotic membrane was then minced into small pieces (4 cm² approximately) and digested twice for 30 min at 37 °C using Trypsin-EDTA 0.25% (Corning, Steuben County, NY, USA) with gentle shaking. For both digestion steps, Trypsin was inactivated with fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA, USA), and the cell suspension was centrifuged for 10 min at 390 g. The cell pellet was resuspended in basal culture medium, Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM H., Corning, Steuben County, NY, USA) containing 10% FBS, 1% penicillin-streptomycin solution, and EGF, 10 ng/mL (Sigma-Aldrich, St. Louis, MO, USA). Single cell suspension was counted and tested for viability using Erythrosin B (Sigma-Aldrich, St. Louis, MO, USA). Only samples with > 90% viability were used for further assays.

Fetal membranes were washed with ice-cold phosphate-buffered saline (PBS, Corning, NY, USA) with 1% penicillin–streptomycin solution (10,000 U/mL penicillin, 10,000 U/mL streptomycin, Corning, Steuben County, NY, USA). The amniotic membrane was mechanically peeled off the underlying chorion layer to remove any blood clots. Then the tissues were incubated for 10 min at room temperature with PBS/ethylenediaminetetraacetic acid (EDTA) 0.5 mM. The amniotic membrane was then minced into small pieces (4 cm² approximately) and digested twice for 30 min at 37 °C using trypsin-EDTA 0.25% (Corning, Steuben County, NY, USA) with gentle shaking. For both digestion steps, trypsin was inactivated with fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA, USA) and the cell suspension was centrifuged for 10 min at 390× g. The cell pellet was resuspended in basal culture medium, Dulbecco’s Modified Eagle’s Medium–high glucose (DMEM H., Corning, Steuben County, NY, USA) containing 10% FBS, 1% penicillin-streptomycin solution, and 10 ng/mL of epithelial growth factor (EGF, Sigma-Aldrich, St. Louis, MO, USA). The single-cell suspension was counted and tested for viability using erythrosin B (Sigma-Aldrich, St. Louis, MO, USA). Only samples with >90% viability were used for further assays.

K₂[PtCl₄], 3-chloro-7-azaindole (3Claza), 3-iodo-7-azaindole (3Iaza), 5-bromo-7-azaindole (5Braza) and solvents (acetone, methanol, ethanol, diethyl ether, N,N′-dimethylformamide, water) were purchased from the following commercial sources - Sigma–Sigma-Aldrich Co. (Praha, Czech Republic), Acros Organics Co. (Pardubice, Czech Republic) and Fisher-Scientific Co. (Pardubice, Czech Republic). The complexes cis-[PtCl₂(3Claza)₂] (1), cis-[PtCl₂(3Iaza)₂] (2) and cis-[PtCl₂(5Braza)₂] (3) (Figure 1) were synthesized by the reactions of water solution of K₂[PtCl₄] with two molar equivalents of naza dissolved in ethanol, and characterized as reported previously [23] (link).
The RPMI 1640 medium and penicillin-streptomycin mixture were purchased from Lonza (Verviers, Belgium). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), phorbol myristate acetate (PMA), Auranofin (98%≤), erythrosin B, and Escherichia coli 0111:B4 lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (Steinheim, Germany). Cell Proliferation Reagent WST-1 was obtained from Roche (Mannheim, Germany). Instant ELISA Kits (eBioscience, Vienna, Austria) were used to evaluate the production of TNFα and IL-1β.

Adherent cells were cultured in the Dulbecco’s modified Eagle medium—DMEM (Gibco, EU) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco, EU), 2 mM glutamine, and 100 U/0.1 mg penicillin/streptomycin. Cells on suspension were cultured in RPMI 1640 (Gibco, EU) medium supplemented with 10 % FBS (Gibco, EU), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES. Cells were grown in humidified atmosphere under the conditions of 37 °C/5% of CO₂ gas in the CO₂ incubator (IGO 150 CELLlife^TM, JOUAN, Thermo Fisher Scientific, Waltham, MA, USA). A erythrosin B (Sigma-Aldrich, St. Louis, MO, USA) dye exclusion method was used to assess cell viability before plating.