Vivid cyp3a4 green screening kit

CYP3A4 and CYP2D6 enzymatic activities were evaluated using a Vivid™ CYP3A4 Green Screening Kit and a Vivid™ CYP2D6 Cyan Screening Kit (both from Thermo Fisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s protocol. According to these assays, the examined compounds are analyzed by their ability to inhibit the production of a fluorescent signal in the reaction between recombinant CYP3A4 or CYP2D6 isozymes and specific Vivid substrates. The experiments were performed in the endpoint mode of the assay, in which solutions of examined compounds were first combined with the Master Pre-mix. After a short incubation, the background fluorescence of the Master Pre-mix and the examined compounds was measured. Next, a mix of the respective Vivid substrate and NADP⁺ was added in order to initiate the enzymatic reaction. After 20 min of incubation, the reaction was terminated using a stop solution and the fluorescence was measured using the recommended (by the manufacturer) excitation and emission wavelengths. Ketoconazole and quinidine (both at 10 µM) were used as positive controls (i.e., inhibitors of CYP3A4 and CYP2D6, respectively). A reaction mixture without tested compounds was used as a negative control and its fluorescence was designated as 100%. The experiments were run in triplicate and the results were presented as means ± SD.

Human antibodies were obtained for TLR4 from BD Pharmingen (San Diego, USA), RIPK1 and MLKL from Proteintech (Rotorua, New Zealand), RIPK3 from Thermo Fisher Scientific (Waltham, USA) and HMGB1 from Abcam (Cambridge, UK). HepaRG cells were purchased from Invitrogen and primary human hepatocytes from ThermoFisher Scientific (Waltham, USA). Acetaminophen (APAP), glycyrrhizin (GL), ethyl pyruvate (EP), necrostatin-1 (Nec-1), tranylcypromine and ketoconazole were purchased from Sigma-Aldrich (Darmstadt, Germany). Dabrafenib was obtained from Selleck. Chemicals (Munich, Germany). Neutralizing IgG monoclonal antibody to human TLR4/MD2, TRIF inhibitory peptide (Pepinh-TRIF) and control peptide (Pepinh-Control) were purchased from InvivoGen (Toulouse, France). To ascertain that used drugs do not interfere with APAP bioactivation, CYP2E1 and CYP3A4 activity were evaluated using Vivid® CYP2E1 Blue Screening Kit and Vivid® CYP3A4 Green Screening Kit (ThermoFisher Scientific, Waltham, MA, USA) (Suppl Figs. 2, 3).