Fc blocker

Isolated microglia samples were first treated with Fc blocker (BD Pharmingen), then stained with surface CX3CR1-APC (1:500, Biolegend) and LIVE/DEAD Fixable Near-IR Dead Cell Staining Kit (1:2000, Invitrogen). The cells were then washed, fixed, and permeabilized with FoxP3/Transcription factor staining set (ThermoFisher), and then stained with Ki67–PerCP–eFluor710 (1:2000, Invitrogen). Flow cytometry was performed with a BD FACS Canto II and the results were analyzed by Flowjo software v10.6.1. Our gating strategy is similar as previously reported⁵⁵ and presented in Supplementary Fig. S3.

After perfused free of blood with PBS, lung, heart, abdominal aorta, hindlimb skeletal muscle, or brain was cut into small pieces, and then incubated with 1 mg/ml collagenase A (Roche Applied Science) for 1 h at 37°C in a shaking water bath (200rpm). After digestion, the tissue was dispersed to a single cell preparation using the gentleMACS™ Dissociator (Miltenyi Biotec) with lung program 2 (which also works well with heart, aorta, skeletal muscle, and brain). The cells were then filtered using a 40 μm Nylon cell strainer and blocked with 20% FBS for 30 min. After 15 min incubation with Fc blocker (1 μg/10⁶ cells, BD Biosciences), the cells were incubated with anti-CD31 (1:1000, BD Biosciences) for 30 min at room temperature. After washing twice, the immunostained cells in 1 ml PBS were added with 50 μl pre-washed Dynabeads conjugated with anti-rat IgG secondary antibody, and incubated for 30 min at room temperature. The cells were then subjected to magnetic purification (Dai et al., 2018 (link)). After washing twice, the cells were used for experiments. Flow cytometry analysis demonstrated that the purity of ECs isolated by magnetic sorting was ~90%. Non-ECs were collected from the wash-through cells after 2 times anti-CD31 incubation and depletion.

Cells were fixed in suspension using 4% PFA at RT for 10min and blocked with 10% goat serum, 3% BSA and Fc blocker (BD Pharmingen, 1:10) in PBS for 30 min at RT. PBMC were incubated with CD45-FITC (eBioscience, 1:20) and TRA-1-60 (Life technologies, 1:50) in blocking at 4 °C overnight and then stained with Alexa Fluor®594 Anti-Mouse (Thermo Fisher, 1:200) for 1 h at RT. The negative control was only incubated with Alexa Fluor®594 Anti-Mouse. Cells were stained with DAPI and mounted onto slides.

B cells were plated on PAA gels and incubated for 15 min at 37 °C. The cells were then transferred to 4 °C and Fc receptors were blocked for 10 min using Fc blocker (BD, 1/200). The cells were washed with PBS and incubated with rabbit anti-HEL antibody at 4 °C for one hour and then with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody for one hour. The cells were moved to room temperature, fixed by incubation with 4% PFA for 10 minutes and permeablized by incubation with PBS plus 0.2% BSA and 0.05% saponin. The cells were then incubated with rabbit anti-HEL antibodies for 1 hour, washed with PBS–BSA-SAPONIN, and incubated with the Alexa 546-conjugated anti-rabbit IgG secondary antibody for one hour at room temperature. The cells were washed several times in PBS and then used for imaging. Images were acquired using laser scanning microscope (Leica) with a × 40 1.4 NA oil immersion objective.