Isotype control mab

LY 294002 and LY 303511 were from Sigma-Aldrich (Rehovot, Israel). Pam3CSK4 was obtained from InvivoGen (San Diego, CA). T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA). Recombinant proteins were from Peprotech (Rehovot, Israel). LysoTracker® Red DND-99 was from ThermoFischer Scientific (MA, USA).

Immature human myeloid DCs from day 6 of culture were harvested, resuspended in fresh DC medium, and exposed to 2 × 10⁵ C. parvum oocysts or to lipopolysaccharide (LPS; Sigma-Aldrich, Darmstadt, Germany) or left without further stimulation (24-well plates, total volume of 500 µL DC medium, 1 × 10⁵ DCs/well overnight). For flow cytometry analysis, differently treated DCs were all harvested on day 7 of culture. Aliquots of approximately 5 × 10⁵ DCs were washed in PBS/2% FCS and incubated for 15 min in 10% heat-inactivated human serum to block Fc receptors. After washing, cells were resuspended in sterile 50 µL PBS/2% FCS with 0.5 µg fluorescence-conjugated monoclonal antibodies (mAbs) directed against the CD surface markers CD1a, CD3, CD14, CD19, CD83, and HLA-DR (BioLegend, Amsterdam, Netherlands) or CD11b, CD58, CD40, and CD86 (ImmunoTools GmbH, Friesoythe, Germany) or with isotype control mAbs labeled with the corresponding fluorescence conjugates (BioLegend, Amsterdam, Netherlands). Some samples were fixed and rendered mAb-permeable using FoxP3^® fixation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) before staining. After incubation for 30 min in the dark, stained cells were washed twice in 1 mL PBS/2% FCS, resuspended after centrifugation in 0.5 mL PBS/2% FCS, and analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

After at least 12 weeks of culture, HSMCs were pre-incubated with anti-Siglec-6 (clone 767329; R&D Systems, Minneapolis, MN, USA) or isotype control mAb (BioLegend, San Diego, CA, USA) at 5 μg/mL for 30 min, unless otherwise indicated, prior to stimulation with the indicated concentration of anti-FcεRIα (clone CRA-1; BioLegend), rhC5a (Peprotech), or compound 48/80 (Sigma-Aldrich). HSMC activation was then assessed by β-hexosaminidase release.