Softmax pro version 5

Human IL-1β protein released into the supernatant was measured using ELISA MAX Deluxe kits (BioLegend), according to the manufacturer’s instructions. Signal from ELISA plates was read with a Spectra Max Plus 384 plate reader (molecular Devices, San Jose, CA) using SoftMax Pro Version 5 software (molecular Devices), and the threshold of detection was 0.5 pg/ml.

Anti-AT1R and anti-ETAR Aabs were detected by using a commercially available sandwich ELISA (One Lambda) as described previously [2 (link)]. Because serum levels can differ from those of purified IgG, we tested Aab levels in IgG fractions after purification. Anti-AT1R and anti-ETAR Aab levels of IgG fractions from healthy donors were usually below 10 arbitrary units, whereas those from SSc patients ranged up to 40 arbitrary units.
Supernatants of treated PBMCs were collected after 8 and 20 hours, respectively, depending on the outcome parameter. Cytokines and soluble proteins were analyzed by performing sandwich ELISAs in a colorimetric detection system with tetramethylbenzidine, hydrogen peroxide and horseradish peroxidase. For the detection of IL-8, renamed chemokine (CXC motif) ligand 8 (CXCL8), the anti-human IL-8 capture antibody H8A5 (Biolegend) and the biotin-coupled detection antibody E8N1 (Biolegend) were used according to the manufacturer’s instructions. To analyze the concentrations of chemokine (C-C motif) ligand 18 (CCL18), transforming growth factor β1 (TGF-β1), soluble CD62 (sCD62) and soluble CD14 (sCD14), duoset kits were used according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was measured in a microplate reader at 450 nm and analyzed using SoftMax Pro version 5 software (Molecular Devices).

An infected mesothelium produces inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and others. Cell-free supernatants were processed for cytokine secretion by enzyme-linked immunoabsorbent assay (ELISA) kits: vascular endothelial growth factor (VEGF), TNF-α, MCP-1, and IL-1β (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at λ450/540 nm with SpectraMax 190 ELISA-Reader and analyzed with Softmax Pro Version 5.0 (Molecular Devices, San José, CA, USA) software. All samples were analyzed twice.

To ensure that the bacterial infection reflected a clinically relevant scenario, cell viability was assessed using a Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol. Absorbance was measured at λ490 nm with SpectraMax 190 ELISA-Reader and analyzed with Softmax Pro Version 5.0 (Molecular Devices, San José, CA, USA). All samples were analyzed in triplicate.

Briefly, Ni²⁺-NTA microplates (Pierce) were incubated with 30 µl/well the supernatant of LASV GPC variants mixed with 70 µl/well of PBS at 4 °C overnight, followed by blocking with 200 μl/well of CelBooster Cell Growth Enhancer Medium for Adherent Cell for 1 h at RT. Between each subsequent step, plates were washed five times with PBS-T (PBS plus 0.05% Tween 20). After plate wash, 100 µl/well of 10 µg/ml GPC-specific antibody 37.7H was incubated at RT for 2 h. followed by incubation for 30 min at RT with 100 μl/well of horseradish perox- idase (HRP) conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories Inc., PA), diluted at 1:10,000 (v/v) in CelBooster Cell Growth Enhancer Medium for Adherent Cell plus 0.02% Tween 20. Then, the reaction signal was developed with 100 μl/well of tetramethylbenzidine substrate (BioFX-TMB, SurModics) for 10 min at RT before the addition of 100 μl/well of 0.5 N sulfuric acid (Fisher Chemical) to stop the reaction. Plates were read at 450 nm wavelength (SpectraMax using SoftMax Pro, version 5, software; Molecular Devices, Sunnyvale, CA), and the optical densities (OD) were analyzed following subtraction of the nonspecific horseradish peroxidase background activity. All samples were measured in duplicate.

Acetylcholinesterase (AChE) activity was measured following a procedure described previously [13 (link)]. Briefly, EV samples (100 µL) were mixed with 1.25mM acetylthiocholine in PBS at pH 8 plus 0.1mM 5,5-dithio-bis (2-nitrobenzoic acid) in PBS at pH 7. The resulting suspension (200 µL) was held at room temperature and then warmed to 37°C for 10 minutes for optical density reading. Absorption at 450 nm was monitored for 10 minutes using a plate reader spectrophotometer (Molecular Devices with SOFTmax^® Pro version 5, San Jose, CA, USA).