α cd40 fitc

The surface marker expression of moDCs was analyzed using flow cytometry. moDCs were seeded in a 24-well plate at a density of 1 × 10⁵ cells per ml, and stimulated with the samples including 1 μg ml⁻¹ of Bet v 1 or 10 μg ml⁻¹ of BPE conjugated to 100 μg ml⁻¹ of SiO₂ NPs for 24 hours. The cells were then washed and stained with α-HLA-DR APC (Invitrogen, Waltham, MA, USA), Fixable Viability Dye eFluor 506 (eBioscience, Waltham, MA, USA), α-CD1a BV421 (Biolegend), α-CD86 PE (eBioscience), α-CD40 FITC (Biolegend), α-CD80 APC-H7 (BD Biosciences, Heidelberg, Germany) and α-CD83 PE-Cy™7 (BD Biosciences) for 30 minutes at 4 °C in the dark. The cells were then fixed with 4% PFA, then suspended in FACS buffer containing PBS and 3 mM EDTA, acquired using an FACS Canto II flow cytometer (BD Biosciences) and analysed using FlowJo X 10.0.7r2 software (BD Biosciences). The cells were washed twice with cold PBS in between the consecutive steps. LPS (100 ng ml⁻¹) was used as the positive control and unstimulated cells were used as the negative control. The gating strategy used for the analysis and the FMO controls are shown in Fig. S13.†

The expression of co-stimulatory molecules was assessed by flow cytometry. moDCs were seeded at a density of 1 × 10⁵ cells per mL in a 24-well plate and incubated with either 1 µg/mL of Bet v 1 bound to 100 µg/mL of SiNP or 100 µg/mL of SiNP without Bet v 1 for 24 h at 37 °C and 5% CO₂. The cells were then collected and stained for α-HLA-DR APC (Invitrogen, Waltham, MA, USA), Fixable Viability Dye eFluor506 (eBioscience, Waltham, MA, USA), α-CD1a BV421 (Biolegend, San Diego, CA, USA), α-CD86 PE (eBioscience), α-CD40 FITC (Biolegend), and α-CD83 PE-Cy™7 (BD Bioscience, Heidelberg, Germany). The cells were then fixed by adding 4% PFA (Sigma) solution in PBS, before sample acquisition using the FACS Canto II flow cytometer (BD Biosciences). The data thus obtained were analyzed using the FlowJo X 10.0.7r2 software. The data are presented in relation to the controls, which has been obtained by excluding the dead cells, nanoparticles, and doublets. The gating strategy is shown in Figure S2, in addition to the fluorescence minus one (FMO) control in Figure S3.