Heat inactivated human ab serum

NK cells were added to monolayers of Vero or A549^ACE2/TMPRSS2 cells, 24 or 48 h p.i. Co-cultures were maintained in LGM-3 medium (Lonza) supplemented with 5% v/v heat-inactivated human AB serum (Sigma), 500 U/ml IL-2 (STEMCELL Technologies), 140 U/ml IL-15 (R&D Systems), and penicillin-streptomycin solution (100 U/ml and 100 µg/ml, respectively) (PAN Biotech GmbH) for 24h. In the case of HAE cultures, NK cells in LGM-3 medium (Lonza) supplemented with 5% v/v heat-inactivated human AB serum (Sigma), 500 U/ml IL-2 (STEMCELL Technologies), 140 U/ml IL-15 (R&D Systems), and penicillin-streptomycin solution (100 U/ml and 100 µg/ml, respectively) (PAN Biotech GmbH) were added to the basolateral side of HAE and maintained for 72 h.

Peripheral blood mononuclear cells (PBMCs) from healthy blood donors (n = 8) were obtained from Sahlgrenska University Hospital. PBMCs from each donor were assayed separately and isolated by density gradient centrifugation using Ficoll‐Paque Plus (GE Healthcare Bio‐Sciences, Uppsala, Sweden). The cells were resuspended in Dulbecco's Modified Eagle's Medium (Invitrogen, Lidingö, Sweden) supplemented with 5% heat‐inactivated human AB serum (Sigma‐Aldrich, Steinheim, Germany), 100 U·ml⁻¹ of penicillin, and 100 μg·ml⁻¹ of streptomycin (Invitrogen). Cell viability was determined by staining with 0.4% trypan blue (Sigma‐Aldrich), and the cells were counted using a Bürker chamber.
PBMCs (2 × 10⁶ cells/per well) were cultured with or without 500 or 1,000 μM HEMA, TEGDMA, EMA, or DEGDA (duplicates) in 24‐well plates and cultured at 37 °C (humidified atmosphere, 5% CO₂) for 24 hr. Cells that were exposed to HEMA, TEGDMA, or EMA had viability levels in the range of 90–95%, whereas more than 50% of the cells that were exposed to DEGDA died.

PBMCs were separated from heparinized blood samples using Ficoll-Paque (GE Healthcare) density gradient centrifugation. PBMCs were cultured in 96-well plates at a concentration of 1 × 10⁶ cells/mL in a final volume of 200 μL of RPMI 1640 medium (Sigma) supplemented with 2 mM L-glutamine (Sigma), 100 U/mL penicillin (Sigma), 100 μg/mL streptomycin (Sigma), and 10% heat-inactivated human AB serum (Sigma). PBMCs were stimulated with SLA (10 μg/mL) for 5 days and lymphoproliferative response was evaluated after adding 1μCi/well of [³H]-thymidine (Amersham, France) for the last 6 h using a liquid scintillation counter (Rack Beta, LKB Wallace, Australia). Results were expressed as Δcpm, difference between mean counts of triplicates in SLA-stimulated PBMC and mean counts of triplicates in unstimulated PBMC.

Viable monocytes were cultured at a density of 1 × 10⁶ cells/mL in 24-well plates in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2% heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), 250 U/mL IL-4 and 200 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (both from Peprotech, London, UK) for 6 days at 37 °C and 5% CO₂ atmosphere. On day 4, culture cell medium and cytokines were refreshed and a maturation cocktail containing 1000 U/mL IL-1β, 1000 U/mL TNF-α (both from Peprotech) and 1 μM prostaglandin-E2 (PGE2) (Pfizer, New York, NY, USA) was added to obtain mDC. For the generation of VitD3-tolDC, monocytes were additionally treated with 1 nM 1α,25-dihydroxyvitamin D3 (Calcitriol, Kern Pharma, Barcelona, Spain) on days 0 and 4. On day 6, cells were harvested by 25 min of accutase incubation at 37 °C to detach the cells from the plate. After washing, cell counts and viability were assessed by flow cytometry as previously mentioned, and phenotype and functional characterization were determined (see below). Dry pellets of each condition with the remaining cells were stored at −80 °C.

Cryopreserved PBMCs from 10 CAVA subjects (with and without α-gal sensitization) were thawed, washed twice with warm complete media (RPMI with L-Glutamine supplemented with 5% heat-inactivated human AB serum (Sigma), 1 mM sodium pyruvate, 0.01 M HEPES, 1x MEM non-essential amino acids, 50 μM 2-Mercaptoethanol, and 1 mM Pen-Strep (all from Gibco). Total B cells were enriched using the STEMCELL human B cell enrichment kit (StemCell Technologies). IgE-expressing B cells were identified and characterized using flow cytometry with mAbs as listed in Supplementary Table 3. Cells were acquired on Cytek Aurora and the analysis was performed using FCS Express 7. The gating strategy is shown in Supplementary Figure 1.

An overnight culture of the yeast was started by inoculating 3 mL of YPD (10 g/L yeast extract, 20 g/L bacteriological peptone, and 20 g/L glucose (Sigma-Aldrich) media, and incubated on a rotor at 20 rpm at 25 °C, prior to conducting the phagocytosis assay. In preparation for phagocytosis, yeast cells were washed in PBS, counted on a haemocytometer, and opsonized with 5% heat-inactivated human AB serum (Sigma-Aldrich). MDMs where then infected with 1x10⁶ yeast cells per well (MOI 10:1), and incubated at 37°C, 5% CO₂ for 2 hours; after which non-engulfed yeast cells were washed away using PBS, and serum-free adhesion media was added to infected MDMs. At 0 (T0) and 18 (T18) hours post-infection, extracellular yeast cells were washed away using PBS and macrophages containing yeast cells were lysed in dH₂O at 37 °C, 5% CO₂ for 30 minutes. For live imaging to quantify vomocytosis, hMDMs were washed at T0, fresh serum-free RPMI added to infection wells and taken for imaging.

Buffy coats from healthy donors were obtained from the Department of Transfusion Medicine and Blood Bank, University Hospital (Brno, Czech Republic). All subjects' blood samples were taken after signing an informed consent form approved by the local ethical committee. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation on Histopaque (Nycomed Pharma, Oslo, Norway). The PBMCs (1 × 10⁶ cells/mL) were placed in a 5 mL Petri dish (Nunclon) for 2 h plastic adherence. To obtain iDCs, adherent cells were cultured in 5 mL of complete media X-VIVO 10 (BioWhittaker, Walkersville, USA) supplemented with 2 mM glutamine (Bio Whittaker), 3% heat inactivated human AB serum (SigmaAldrich, USA), 800 UI/mL GM-CSF (PeproTech, USA), and 500 UI/mL IL-4 (Prospec, Israel) for 6 days, media was changed after 3 days. On day 6, iDCs were matured for a further 6 h or 24 h by 50 ng/mL IFN-γ (ProSpec, Israel) and 200 ng/mL LPS (Calbiochem, MA, USA). The resulting DCs were called aDCs. tDCs were matured using 1 ng/mL IL-10 (PeproTech, USA) and 2 ng/mL TGF-β (PeproTech, USA) for the same time period as the aDCs. The DCs measurements and harvests were carried out on day 6 of cultivation (0 h) and at 6 h and 24 h of maturation.