Mojosort streptavidin nanobeads

For all mice in vivo experiments, BAL were obtained by flushing the lungs three times with 1 mL PBS containing 0.5 mM EDTA (Sigma-Aldrich). Human PBMCs were isolated from asthma patient blood draws or buffy coats as previously described^{65 (link)}. Red blood cells were lysed using osmotic lysis buffer (8.3% NH₄Cl, 1% KHCO₃, and 0.04% NA₂EDTA in Milli-Q water). To purify human CD8 T cells for cultures, PBMCs from buffy coats were first depleted for CD4⁺, CD14⁺, CD16⁺, CD19⁺_, CD36⁺, and CD235ab⁺ cells by magnetic cell sorting (MACS) using MojoSortTM streptavidin Nanobeads (BioLegend, USA) and LS Columns (Miltenyi Biotec). After MACS, cells were rested overnight in RPMI-1640 medium supplemented with 5% fetal calf serum (FCS) at 4 °C. The next morning, cells were extracellularly stained with antibodies for 30 mins at 4 °C and with LIVE/DEADTM Fixable aqua for 15 mins at 4 °C. CD8 T cells were sorted by fluorescence-activated cell sorting (FACS) using a FACSAriaTM III (BD).

Mouse ears were separated into two skin halves and treated with 0.5% trypsin (Alfa Aeser, Ward Hill, MA) at 37 °C for 30 min. Epidermis and dermis were separated and dermis was digested with collagenase IV (1–2 mg ml⁻¹, Worthington, Lakewood, NJ). Epidermal cells were released after trypsin digestion without using collagenase treatment. The trunk skin of neonatal mice was similarly processed to obtain single cell suspensions. For qRT-PCR analysis of Rara and Cd207 genes, CD11c⁺ cells from BM culture or CD11c⁺ cells and CD45-negative tissue cells from single suspensions of epidermis or dermis were isolated by magnetic selection with biotin-labeled CD11c (clone N418) and Mojosort^TM streptavidin nanobeads (BioLegend; purity > 90%). Gradient centrifugation of epidermal and dermal cells released by collagenase digestion was performed on a 40/70% Percoll gradient to enrich leukocytes.

The BM cells were blocked with TruStain fcX (anti-mouse CD16/32) antibody (Clone 93; BioLegend, San Diego, CA, USA) and stained with Biotin anti-mouse Ly-6G antibody (Clone 1A8; BioLegend) for 15 min at 4 °C. The cells were then washed and resuspended in 2% FBS/PBS and separated into Ly-6G⁺ and Ly-6G⁻ BM using a MojoSort magnetic cell separation system and MojoSort streptavidin nanobeads (BioLegend) following the manufacturer's instructions.

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.