Mojosort streptavidin nanobeads

Intestinal tissue was washed with Hank’s balanced salt solution (HBSS, Biosharp) and then treated first with HBSS containing 1 mM EDTA (Sigma-Aldrich) for 20 min at 37 °C, followed by collagenase type IV (STEMCELL Technologies) for 2 h at 37 °C. The cell suspension was incubated at 4 °C for 30 min with an anti-rat CD68 antibody (biotinylated, Supplementary Table 1). Afterward, MojoSort Streptavidin Nanobeads (BioLegend, San Diego, CA, USA) were added to the cell suspension and incubated at 4 °C for 15 min. CD68+ cells were isolated by magnetic cell sorting.

P14, Mini or Maxi CD8 T cells were enriched from spleens and LNs by negative selection using the mojosort CD8 T cell isolation kit according to manufacturer’s protocol (Biolegend). 10⁴ (unless otherwise stated) TCR transgenic cells were adoptively transferred into new hosts that were subsequently infected with MCMV. After at least 4 weeks, Tcf1⁺ and Tcf1⁻ cells were sorted from spleens and LNs, using a BD FACS Aria sorter. CD4 T cells and B cells were depleted before cell sorting, using biotinilated CD4 and B220 antibodies and Mojosort Streptavidin nanobeads (Biolegend).

Peripheral blood mononuclear cells from buffy coat were enriched for ILCs using a negative selection protocol for CD3, CD14, CD16, and CD19 using Mojosort Streptavidin Nanobeads (Biolegend) as described by Krabbendam et al. (41 (link)). CD5^-CD7⁺ and CD5⁺CD7⁺ ILCs were then purified from CD127⁺Lin^-CD3^-TCRαβ^-CD94^- cells using a MA900 cell sorter (Sony Biotechnology). Each ILC subset (1,000 cells per well) was co-cultured with OP9-DL1 cells (3,000 per well) in a 96-well U-bottom plate. OP9-DL1 cells were maintained in IMDM/10% FCS with penicillin/streptomycin before co-culture with ILCs. Cells were co-cultured for one week in IMDM supplemented with Yssel’s media and 2% human serum, together with 20 ng/ml IL-2 and 20 ng/ml IL-7 (Miltenyi Biotech). For polarizing conditions the following cytokines (Peprotech) were added (all at 20 ng/ml unless stated otherwise) (1) ILC1s: IL-1β and IL-12; (2) ILC2s: IL-4, IL-25, IL-33, and TSLP; (3) ILC3s: IL-1β and IL-23. Cytokines were replenished at day 5 of culture. At day 7 of culture, cells were harvested, stimulated with PMA and ionomycin and stained for intracellular cytokines as described above.

For uptake assay for apoptotic neutrophils, splenic neutrophils were first isolated from the spleen of BALB/c wild-type mice by magnetic sorting using biotinylated anti-Ly6G antibody (clone: 1A8; catalog#: 127604; BioLegend) and Mojosort streptavidin nanobeads (BioLegend, catalog#: 480016). Apoptosis of Ly6G⁺ neutrophils was then induced by incubating neutrophils for 48 h in RPMI complete medium, followed by labeling with AcidiFluor orange-NHS (Goryo chemical). 1 × 10⁶ cells of apoptotic neutrophils labeled or unlabeled with AcidiFluor were administered to IgE-CAI skin lesion on day 5 post-challenge. For uptake assay for OVA, 100 μg of OVA labeled or unlabeled with AcidiFluor dye (Goryo chemical) were administered to IgE-CAI skin lesion on day 5 post-challenge. Ear skins were subjected to flow cytometric analysis 2 h after administration of apoptotic neutrophils or OVA.

Cells were cultured in RPMI complete medium (RPMI 1640 medium (Nacalai tesque) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin (Nacalai tesque), 100 μg/mL streptomycin (Nacalai tesque), 1 mM sodium pyruvate (Nacalai tesque), 0.1 mM nonessential amino acids (Nacalai tesque), and 5 × 10⁻⁵ M 2-mercaptoethanol (Gibco)). Bone marrow-derived basophils (BMBAs) were generated by culturing bone marrow cells in the presence of murine IL-3 (300 pg/mL; BioLegend) for 6 days^{49 (link)}. BMBAs were then sensitized with anti-TNP IgE antibody (clone: IgE-Lb4: 1 μg/mL) for 24 h, followed by the magnetic enrichment of CD49b⁺ fractions by using biotinylated CD49b antibody (clone: DX5, catalog#: 108904, dilution 1:400; BioLegend) and MojoSort Streptavidin Nanobeads (BioLegend). BMBAs were then incubated with TNP-OVA or control OVA (10 ng/mL each) for 18 h. Ly6C^hi monocytes isolated from the bone marrow of BALB/c mice or Il4ra^−/−mice were incubated for 24 h or 48 h with the culture supernatant of BMBAs. In some experiments, Ly6C^hi monocytes (1 × 10⁵ cells/well) from WT mice were incubated for 24 h or 48 h with recombinant mouse IL-4 (20 ng/mL; BioLegend), apoptotic neutrophils (1 × 10⁶ cells/well) or control PBS.