Nucblue live cell readyprobes reagent

Human neutrophils were cultured under the designated condition for 5 h. Hoechst33342 (2 drops/mL; NucBlue Live Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA) and SytoxOrange (1:1000; Invitrogen Inc., CA, USA) were added to the culture medium. Neutrophils were then observed using the EVOS FL Cell Imaging System (Life technologies, CA, USA).

Human neutrophils cultured under the designated condition for 6 h were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with PBS containing 0.2% Tween20 for 10 min. After washing with PBS three times and incubating for 20 min in the SuperBlock (PBS) Blocking Buffer (ThermoFisher Scientific, MA, USA), cells were treated with a rabbit anti-LL-37 antibody (1:1000; OSC00009W, Osenses Pty Ltd., Australia) for 1 h at room temperature. After washing with PBS containing 0.1% Tween20 three times, LL-37 was labeled with Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:1000; Invitrogen Inc., CA, USA) for 1 h at room temperature. After an additional three washes in PBS containing 0.1% Tween20, DNA was stained with DAPI (NucBlue Fixed Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA). Slides were mounted in ProLong Gold antifade reagent (ThermoFisher Scientific, MA, USA). Images were acquired with a Zeiss LSM700 confocal microscope (Carl Zeiss, Germany). Separately, for staining of live/dead neutrophils and NETs, Hoechst33342 (2 drops per mL; NucBlue Live Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA) and SytoxOrange (1:1000; Invitrogen Inc., CA, USA) were added into the culture medium 6 h after initiation of treatment. Neutrophils were then observed with EVOS FL Cell Imaging System (Life technologies, CA, USA).

Human iPSCs (CGT-RCiB10, Cell and Gene Therapy Catapult) were differentiated into iHEPs essentially as described^{44 (link)}, except for the Matrigel (Corning) sandwich and addition of forskolin (5 μM; BioGems) from day 17. On day 19 hepatocyte growth factor was removed and taurine was added (58.4 mg/L; Sigma) and the cells are placed in normoxia. On day 25 the iHEPs were treated +/−20 nM staurosporine (Generon) for 2 h followed by addition of the MRP2 transport substrate precursor CDFDA (10 µM; Sigma-Aldrich) and NucBlue Live Cell ReadyProbes Reagent (Thermo Fisher Scientific) and incubated for a further 15 min at 37 °C. CDFDA is cleaved by intracellular esterases to yield fluorescent 5-(and-6)-carboxy-2′,7′–dichlorofluorescein (CDF; green) which is secreted into the bile canaliculi by MRP2. Cell nuclei and accumulation of CDF (green) in bile canaliculi were captured in images taken on an LSM 880 confocal microscope (ZEISS). Images were analysed using Fiji^{45 (link)}. Images were first processed using global thresholding to filter out noise. Then, size-exclusion filtering was applied to remove particles <5 µm² to select biologically relevant signals to determine integrated density of CDF staining in the bile canaliculi. The statistical significance of experimental data was assessed by Student’s test.