Ensight plate reader

Three-dimensional spheroids were generated and cultured in the InSphero GravityTRAP TM ULA 96-well plates (PerkinElmer) according to the manufacturer’s instructions. These plates were conically shaped, flat-bottom, clear 96-well polystyrene microplates, facilitating the formation of spheroids after incubating seeded cells. In addition, plates were coated to turn them into an ultr-low-attachment (‘ULA’) surface, preventing mammalian cell adherence. Five hundred cells per well were seeded, and spheroid maturation occurred within 1 or 2 d of seeding. Every 2 d the medium was changed. Spheroid growth was measured for up to 15 d after initiation of treatment (day 0) with 2–3 d intervals. Between measurements, the microtissues were incubated at 37 °C and 5% CO₂. Spheroids were imaged using a 4× objective on the image module of an EnSight TM plate reader (PerkinElmer), and both brightfield and mCherry channels were set with NIR LED and True GREEN LED respectively. Acquired Images were automatically analyzed by Kaleido 2.0 software based on detection masks generated with a custom-developed algorithm that automatically identified and measured spheroid areas.

To determine the signaling profile of Nb29 against α_1AAR, we used a cAMP Glo-sensor kit (Promega) with an engineered Gsq protein in which 15 residues at the C-terminus of Gs protein were replaced with those of Gq protein. The Gsq protein could be activated by the α_1AAR and stimulates intracellular cAMP production. In brief, pGloSensor™−22F plasmid, receptor plasmid, and Gsq plasmid were transfected into HEK293T cells. At 24 h after transfection, the cells were switched into a CO₂-Independent Medium (Gibco) and incubated with GloSensor^TM cAMP reagent. The mixture was then transferred to a 96-well white plate. The 96-well plate is placed at 37 °C in the dark for 1 h, then placed at room temperature in the dark for 1 h before use. The luminescence signal was measured by Ensight^TM plate reader (PerkinElmer) around 10–15 min after the addition of the agonist and/or Nb29. The result curves were calculated and fitted by GraphPad Prism 9.

Cell conditioned medium was collected every other day starting from dd5 of iDE and subsequently from all culture conditions. The presence of alpha-fetoprotein (AFP) was quantified using a Cobas analyzer (Roche, Basel, Switzerland). Human alpha-1 antitrypsin (AAT) was quantified with a sandwich ELISA. Further, 96-well half-volume high-binding plates (Corning) were coated overnight with 2 μg/mL polyclonal anti-mouse IgG (Sigma), saturated for 1 h at 37 °C with blocking buffer (PBS, 0.25% BSA, 0.05% Tween-20) followed by incubation with conditioned medium of anti-human α1 antitrypsin (AAT) monoclonal antibody 3C11 hybridoma cells, which were kindly provided by Prof. David Lomas (University College London, UK) [13 (link),14 (link)]. After washing with PBS/0.05% Tween-20 (PBS-T), serial dilutions of the purified plasma AAT standard and of the cell culture media were added to the plates and incubated at 37 °C for 1 h. After washing with PBS-T, the wells were incubated for 1 h at 37 °C with sheep anti-AAT-HRP-conjugated antibody (Abcam, Cambridge, UK) diluted in blocking buffer, further washed, and revealed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Merck). The reaction was blocked by adding 3 M HCl, and absorbance at 450 nm was measured using an EnSightTM plate reader (PerkinElmer, Waltham, MA, USA).