Easysep magnet

Cell subpopulations of CD4⁺ T cells (CD3⁺CD4⁺) and monocytes (CD3⁻CD14⁺) were isolated using EasySep magnets (STEMCELL Technologies, Vancouver, British Columbia, Canada). First, PBMCs were washed in FACS buffer (PBS + 2% FBS + 2 mmol/l EDTA) and treated with DNAse (100 ug/ml) (StemCell, #7900) for 15 min at room temperature. Subsequently, Easysep Human CD4⁺ positive selection kit II (StemCell, #17852) was used according to the manufacturer's protocol, and the flowthrough used for Easysep Human monocyte isolation kit (StemCell, #19359), according to the manufacturer's protocol.

For macrophage phagocytosis experiment, lung tissue from 2-week tumor-bearing mice and naive mice as obtained and digested into single cell suspension described above. Then cell suspension was stained with fluorochrome-conjugated anti-mouse monoclonal Abs specific for Biotin anti-mouse F4/80 (clone BM8) at 4°C for 15 min in dark. After washed with cell staining buffer and centrifuged at 300 g for 5 min, MojoSort™ Streptavidin Nanobeads were added into 100μl cell suspension and incubated at 4°C for 15 min in dark. After second wash step and positive magnetic sorting was operated by EasySep Magnets (STEMCELL) according to the manufacturer’s instructions.
Then macrophage was seeded into 12 wells plate and Fluorescent yellow-green Latex beads (Sigma-Aldrich, #L4655) were added in the wells and gently shaking on the Transference Shaker. After 30min, the plate was washed with PBS softly to exclude extra beads and analyzed in LSM 710 confocal microscope (Carl Zeiss, Germany).

UCB samples were obtained from the Blood and Tissue Research Biobank (Barcelona, Spain). All samples were obtained between 20 and 72 h after delivery. To isolate CD34⁺ cells, UCB were incubated with RosetteSep (STEMCELL Technologies, Vancouver, Canada) for 20 min prior to density gradient purification using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and centrifugation at 700g for 30 min. CD34⁺ cells were selected from the mononuclear cells fraction using the EasySep human cord blood CD34⁺ selection kit II (STEMCELL Technologies, Vancouver, Canada). Briefly, mononuclear cells were incubated with anti-human CD34 antibodies and FcR-blocking antibodies for 10 min. Then, dextran microbeads were added to the cell suspension and incubated for 1 min at room temperature. The immunomagnetic selection was performed using EasySep Magnets (STEMCELL Technologies) according to the manufacturer’s instructions. Purity, viability, and cell number were then assessed by flow cytometry using anti-human CD34-PE (BD Biosciences, San Jose, CA, USA), anti-human CD45-FITC (BD Biosciences), 7-amino-actinomycin-D (7-AAD, BD Biosciences) and Perfect Count Microbeads (Cytognos, Salamanca, Spain). Only samples with cell purity higher than 75% and viability higher than 90% were used.

Thymocytes from WT mice were enriched for Vα14i NKT cells by magnetic depletion using biotinylated antibodies against CD8α, CD19, CD24, and TER-119 (BD Biosciences and eBioscience) together with EasySep magnets and protocols and reagents from StemCell Technologies. Cells were then stained with αGalCer-loaded CD1d tetramers, together with anti-TCRβ antibodies, in staining buffer containing 1 μg/ml streptavidin. Tetramer-positive, TCRβ⁺ cells were isolated using a FACSAria cell sorter (BD Biosciences). Sorted Vα14i NKT thymocytes were then cultured for 48 h at 10⁶/ml in complete RPMI media on a plate coated with anti-TCRβ antibody together with soluble anti-CD28. Cells were then maintained by culture in complete RPMI media with 10 ng/ml mouse IL-15/IL15Ra (eBioscience) for 5 days before being used in experiments.

Bone marrow lineage⁻c-Kit⁺ (LK) HSPCs were separated by EasySep magnets (cat #: 18000, STEMCELL Technologies, Vancouver, BC, Canada) using lineage-negative and c-Kit-positive selection kits (cat #s: 19856 and 18757, STEMCELL Technologies, Vancouver, BC, Canada). To determine the effects of PGN on HSPCs ex vivo, the cells were pretreated with purified PGN (100 μg/mL), for 2 h in culture medium [12 (link)].

CD4⁺CD25^– T cells, isolated by MACS from freshly separated PBMCs using EASYSEP Magnets (Stemcell Technologies) by the manufacturer’s protocol, were cultured in complete medium (Corning RPMI 1640) with 10% pooled human AB serum (GemCell U.S. Origin Human Serum AB), 1% L-glutamine, and 1% penicillin-streptomycin. Functional-grade purified anti–human CD3 (OKT3) and functional-grade purified anti–human CD28 (CD28.2) (both from eBioscience) were added at 2 μg/mL. Recombinant IL-2 (200 ng/mL, Peprotech), TGF-β (5 ng/mL), ACTH (100-1000 ng/mL, MilliporeSigma), and MC2R blocker (400 nM/mL, MyBioSource) were also added.

CD4⁺CD25^– cells were isolated from the spleens of mice by MACS using EASYSEP Magnets (Stemcell Technologies), according to the manufacturer’s protocol and placed in a 96-well plate. Tregs were induced by addition of anti–mouse CD3 (clone 145-2C11, eBioscience) and CD28 (clone 37.51, eBioscience) antibodies (3 μg/mL in complete RPMI media), TGF-β (10 ng/mL, BioLegend) and IL-2 (100 ng/mL, BioLegend) cytokines, and ACTH (100ng/mL, MilliporeSigma). The plate was incubated in 5% CO₂ at 37°C for 3 days.