Ultra low attachment plate

To assess the effects of IFX on NETosis generation, HD neutrophils (n = 3) were isolated and pretreated for 15 min at 4 °C with FCRII blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, neutrophils were washed with PBS, centrifuged at 300 g for 10 min, seeded in 24 well plates on poly-L lysine-coated glass coverslips (BD Biosciences) (6 × 10⁵ cells per well) in RPMI 1640 supplemented with 20% BSA (PanReacAppliChem, Barcelona, Spain), 2 mM L-glutamine (Biowest, Nuaillé, France) and 1% ZellShield (Minerva Biolabs, BMBH, Berlin, Germany) at 37 °C and 5% CO₂. Next, neutrophils were treated with TNF-α (8 ng/mL; PrepoTech, Rocky Hill, NJ, USA), a potent NETosis inducer, in the presence or in the absence of IFX (100 mg/ml; Pfizer, NY, USA) for 2 h. NETotic response was evaluated as previously described.
To evaluate the effects of IFX on the proinflammatory profile of mononuclear cells promoted by NETs, PBMCs from previous HDs (n = 3) were seeded in 24-well ultra-low attachment plate (Sigma Aldrich) (1 × 10⁶ cells per well) and treated for 24 h with supernatants obtained from above-described in vitro experiments which were pre-incubated in the presence or absence of 15 U/ml DNAse I (NZYTech-Genes & Enzymes, Lisbon, Portugal) for 10 min. PBMCs were harvested for protein and RNA determination.

Forebrain specific cerebral organoids were produced using the previously published protocol (Qian et al., 2016 (link)) with some alterations. At day 0, hESCs were passaged with Accutase (Sigma-Aldrich) and cells were resuspended in mTeSR 1 or Plus containing Y-27632. A cytometer was used to estimate the number of cells in the single cell suspension. The single cell suspension was added to pretreated wells AggreWell 800 (Stem Cell Technologies) that would result in 5000 cells per microwell. Pretreatment was performed with Anti-Adherence Rinsing Solution (Stem Cell Technologies) and handling of the AggreWell 800 plates was carried out following the manufacturer’s recommendations. At day 1, embryoid bodies were harvested from the AggreWells per the manufacturer’s protocol and transferred to a well of a 6 well Ultra-Low Attachment Plate (Sigma-Aldrich) containing mTeSR-E5 (Stem Cell Technologies) with Dorsomorphin (2μM, Sigma-Aldrich) and A83-01 (2μM, Tocris). Embryoid bodies were maintained in this medium until day 4, after which the protocol described (Qian et al., 2016 (link)) was followed. Organoids were either dissociated using Accutase (Sigma-Aldrich) at 37°C for 30min for FACS analyses or fixed for 30min in Paraformaldehyde (PFA, 4%, Sigma-Aldrich) and were stored in sucrose (30%, Sigma-Aldrich) until further use.

To construct the cardiac organoids, enriched day-19 iPSC-derived cardiomyocytes were seeded onto Matrigel-coated 48-well Nunc^TM UpCell plates at 2 × 10⁵ cells/cm² in DMEM/F-12 GlutaMAX media supplemented with 20% fetal bovine serum (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 50 U/mL penicillin/streptomycin and 10 μM Y − 27632. After 24 h, iPSC-derived endothelial cells (8.6 × 10⁴ cells/cm²) were then seeded onto the iPSC-cardiomyocytes and cultured in cardiac organoid medium consisting of a mixture of cardiomyocyte media and EGM2-MV media supplemented with 50 ng/mL VEGF-165 (at 1:1 ratio). After 24 h, the UpCell plates were brought to room temperature and the detached cell sheet was transferred to an ultralow attachment plate (Sigma-Aldrich) containing cardiac organoid media for 24 h. The resulting spheroids were then embedded in 15 µL of growth factor reduced Matrigel and cultured in cardiac organoid media. Cardiac organoids were maintained in culture in a humidified CO₂ incubator on an orbital shaker at 100 rpm and media was changed every 2–3 days.