Dq ovalbumin

2 × 10⁵ cDC1 from day-11-Flt3L-cultured BM cells were incubated with β2m^−/− splenocytes osmotically loaded with 10 μg/ml DQ-Ovalbumin (Cat#D12053; Invitrogen) in the presence or absence of MG132 (Cat# HY-13259; MCE) for different times. Then cDC1 were washed with PBS for three times and stained with antibodies against cDC1 markers before analysis for FITC⁺ cells.

Endotoxin-free ovalbumin (1 mg/mL, Hyglos) was opsonized with a polyclonal rabbit anti-ovalbumin antibody (3.7 mg/mL, Sigma-Aldrich) at 37°C for 1 hour, at a 1:10 (v/v) ratio, to form insoluble immune complexes (IC), for stimulation of cells in culture or administration in vivo. Cells were harvested at defined time points and washed to remove free ICs. For RNA sequencing experiments, BMDMs and BMDCs were stimulated with immune complexes of ovalbumin for 4 hours. For phagocytosis assays, Alexa Fluor (AF)647-conjugated ovalbumin was used (Thermo Fisher), and conjugated with antibody as described above. For in vivo phagocytosis, 0.33 g/kg AF647-conjugated ovalbumin was opsonized with 3.2 g/kg polyclonal rabbit anti-ovalbumin antibody before injection. Phagocytic degradation products were identified by substituting Ova-647 with DQ^™ Ovalbumin (Invitrogen), a conjugate that exhibits fluorescence only after proteolytic degradation.

Lysosomal Intracellular Activity Assay Kit (BioVision, #K448) was initially used for assessment of lysosomal cleavage of a self-quenching substrate. Subsequently, most of experiments used DQ ovalbumin (10 µg/mL, Invitrogen, #D12053), which was added into each cell suspension (1 mL per sample) and incubate at 37 °C for 30 min. Cells were collected and washed with PBS once and then stained with flow antibodies of DC markers (anti-CD45, anti-CD11c and anti-MHC II Abs). Lysosome activity was detected by FACS using 488 nm excitation laser (515/20 Blue channel of LSRFortesssa flow cytometer).

The proteolytic degradation in lysosomes was evaluated using DQ ovalbumin (Invitrogen, D12053) as previously described.²¹, ²³ For the isolation of primary TECs, kidneys were retrieved and washed with saline. The renal cortices were then minced into tiny particles by cutting with scissors into 1 mL of DMEM medium and subjected to digestion by addition of 10 μL Liberase (Roche, 05401020001) in a water bath at 37°C for 20‐30 min with pipetting every 5 min. Thereafter, 1 mL of DMEM supplemented with 10% FBS was added to stop the digestion and transferred onto a pre‐wetted 70‐μm cell strainer (Biologix, cat# 15‐1070). The filtrate was collected and centrifuged at 200 g and 4°C for 5 min. After re‐suspension in 1 mL of DMEM, approximately 2 × 10⁵ primary TECs were added to a six‐well plate and then incubated with 1 µL anti‐Cadherin‐16 antibody (NOVUS, NBP2‐47745) and 2 µg/mL DQ ovalbumin for 2 h at 37°C in the dark. For the in vitro experiment, HK‐2 cells were incubated with 2 µg/mL DQ ovalbumin for 2 h at 37°C in the dark. The mean fluorescent intensity of DQ ovalbumin was quantified by flow cytometry.

HK-2 cells were incubated with 10 μg/mL DQ-ovalbumin (Invitrogen, Carlsbad, CA, United States) for an additional 2 h at 37°C. Then the cells were washed with PBS and fixed with 4% paraformaldehyde. The mean fluorescence intensity of green fluorescent DQ-ovalbumin puncta in individual podocytes was calculated and presented in the figures.