Tristar multimode reader lb942

Feces were suspended in 10 times weight/volume (w/v) of PBS. After centrifugation (8000 × g for 15 min), supernatants were collected as fecal extracts. Plates were pre-coated with 1 mg ml⁻¹ OVA overnight, followed by blocking for 1 h at room temperature with PBS containing 1% (w/v) bovine serum albumin. Then fecal extracts with serial dilutions were added for incubation for 1 h at room temperature. The relative binding ability of IgA was detected with alkaline phosphatase-conjugated goat anti-mouse IgA (Southern Biotech, USA). After incubation at 4°C overnight, the OD values at 405 nm were measured with a TriStar Multimode Reader LB 942 (BERTHOLD, Germany).

Metabolic activity of mature HepaRG cells was determined using the XTT assay according to the manufacturer’s instructions (AppliChem). Briefly, XTT reagent (1 mg/mL) was added on cell-laden 3D constructs and incubated for 4 h (37 °C, 5% CO₂). The absorbance of the supernatant was measured spectrophotometrically at A450 nm (TriStar Multimode Reader LB942, Berthold Technologies, Bad Wildbad, Germany) with a reference of A620 nm.
Lactate dehydrogenase (LDH) release of mature HepaRG cells was measured with the LDH detection kit (Roche, Grenzach, Germany), according to the manufacturer’s instructions, in the supernatant at an absorbance of A492 nm with a reference of A620 nm (Sunrise absorbance microplate reader, Tecan). XTT and LDH values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.
The LIVE/DEAD assay of mature HepaRG cells was performed with the Viability/Cytotoxicity kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, cell-laden 3D constructs were stained with 2 µM calcein-AM and 2 µM ethidium homodimer-1 diluted in 1x Hank’s balanced salt solution (HBSS; Thermo Fisher Scientific) for 30 min (37 °C, 5% CO₂). The stained cell-laden 3D constructs were analyzed by fluorescence microscopy (Zeiss Observer. Z1 microscope).

Concentrations of human interleukin 29 (IL-29/IFN-λ₁) in supernatants of infected cell-laden 3D printed constructs were determined with the IL-29 Human ELISA Kit (Thermo Fisher Scientific, USA) at A450 nm (TriStar Multimode Reader LB942, Berthold Technologies, Germany) with a reference of A620 nm.