Magnisort beads

Antigen-specific naive CD8⁺ T cells were isolated from the spleen of P14 TCR-transgenic mice and purified by negative selection using magnetic-activated cell sorting (Miltenyi Biotec). T cells were then labeled with 5 μM CellTrace™ Violet (Thermo Fisher) and cocultured for 3 days with either BMDCs or SPDCs at a ratio of 5:1 (500,000 T cells:100,000 DCs). For stimulation, 20 ng/ml GP_33-41 peptide and GolgiPlug/GolgiStop™ (BD Bioscience) were added to the cultured cells for the last 5 hours. CD8⁺ OT-I T cells were isolated from the spleen of OT-I transgenic mice and purified by negative selection with MagniSort™ beads (Thermo Fisher). The T cells were then labeled with 5 μM CellTrace™ Violet (Thermo Fisher) and cocultured for 3 days with either BMDCs or SPDCs pulsed for 2 hours with the OVA protein (20 μg/ml) at a ratio of 5:1 (250,000 T cells:50,000 DCs). For stimulation, 0.5 μg/ml OVA_257-264 SIINFEKL peptide and GolgiPlug/GolgiStop™ (BD Bioscience) were added to the cultured cells for the last 5 hours.

BM suspensions were prepared as described above, and lineage-positive cells were depleted using a lineage depletion kit (eBioscience mouse hematopoietic lineage biotin panel; ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, BM derived from the femurs and tibiae of three mice was incubated with biotinylated antibodies against CD3, B220, Gr1, and Ter119 in a volume of 1 ml PBS/0.1% BSA for 10 min at 4°C. Cells were then washed and incubated with 0.4 ml streptavidin bound to magnetic beads (Magnisort beads; ThermoFisher Scientific) in a volume of 2 ml PBS/0.1% BSA in a FACS tube for 10 min at room temperature. The volume was then made up to 4 ml, and the FACS tube was placed inside a magnet for 10 min at room temperature. The fraction of cells not bound to the magnet was poured out and stained for FACS as described above.
Pre–CFU-Es and CFU-Es were sorted by FACS using a FACS ARIA III (BD) with 70-µm nozzle. For culture, cells were sorted into 0.5 ml IMDM medium with 5% FCS in a 5-ml FACS tube. For RT quantitative PCR (RT-qPCR), cells were sorted into 350 µl RLT buffer with β-mercaptoethanol in 1.5 ml tubes. The following staining panel was used for sorting: Sca-1-Pacific blue, PE-CD117, APC-CD105, PerCP Cy5.5-CD11b and Streptavidin (BioLegend), APC Cy7-CD16/32, PE Cy7-CD41, BV605-CD150.