Pe selection cocktail

HSCs were collected according to the manufacturer's protocol for the magnetic bead‐based EasySep Mouse SCA1 Positive Selection Kit (StemCell Technologies, Vancouver, British Columbia, Canada). Briefly, bone cells were collected from the femur and tibia of C57BL/6 mice and made into a suspension of 1×10⁶ cells mL⁻¹ in StemSpan Serum‐Free Expansion Medium (SFEM) (StemCell Technologies) containing human IL‐6 (100 ng mL⁻¹, StemCell Technologies, Canada), human fms‐like tyrosine kinase‐3 (Flt3) ligand (100 ng mL⁻¹, StemCell Technologies), murine stem‐cell factor (SCF) (50 ng mL⁻¹, StemCell Technologies), and human thrombopoietin (TPO) (20 ng mL⁻¹, PeproTech, Cranbury, New Jersey, USA). Mouse SCA1 PE Labeling Reagent (50 µL mL⁻¹, StemCell Technologies) was first added to the cell suspension, and the mixture was kept in the dark at room temperature for 15 min. Then, a PE Selection Cocktail (70 µL mL⁻¹, StemCell Technologies) was added to the mixture and kept in the dark at room temperature for 15 min. After vortexing, dextran RapidSphere (50 µL mL⁻¹, StemCell Technologies) was added to the mixture and stored at room temperature in the dark for 10 min. After the mixture was incubated in the magnet for 5 min, the supernatant was discarded and SFEM was added to resuspend the cells. This step was repeated three times to obtain HSCs, and the cells were used in passages 3–5.

Bone marrow cells were isolated from the tibia and femur and cultured in RPMI1640 medium with 10% FBS, 1% penicillin‐streptomycin, 55 µm β‐mercaptoethanol and 10% L929 conditioned media containing macrophage‐colony stimulating factor (M‐CSF) for 6 days to harvest BMDMs. Mouse T cells were isolated from spleens by using the Dynabeads Untouched Mouse T Cells Kit (Thermo Fisher). CD11b⁺Ly6C^hiLy6G^– cells were sorted on a MoFlo Astrios instrument. For CD11b⁺Ly6G⁺ isolation, cells were labeled with biotinylated anti‐Ly6G (BioLegend), incubated with streptavidin microbeads (BD), and separated on magnetic columns (Stemcell). For specific cell isolation from splenocytes, pDCs were isolated using anti‐mPDCA‐1 microbeads from Miltenyi Biotec (Auburn, CA). After pDCs isolation, macrophages were isolated with CD11b microbeads from Miltenyi Biotec, cDCs were isolated with mouse CD11c PE labeling and followed by PE selection cocktail from STEMCELL technologies, following the manufacturer's protocol. RNAs from pDCs, cDCs, and macrophages were isolated using TRIzol reagent (Invitrogen) and subjected to semi‐quantitative PCR analysis of 18S rRNA by using specific primer.