Aria 3 cell sorter

To generate a single-cell suspension from the skin, subcutaneous fat was removed, and the skin was chopped into 1-mm pieces which were then incubated in liberase (300 µg/mL; Cat# 5,401,119,001, Sigma-Aldrich, Darmstadt, Germany) and DNase 1 digestion cocktail (50 U/mL, Cat# Roche-11284932001, Sigma-Aldrich, Darmstadt, Germany) for 60–90 min at 37 °C as described [28 (link)]. Digested skin cells were passed through a 100-µm pore filter and then through a 40-µm pore filter. The single cells obtained were washed once in 1% BSA and then blocked with FcR blocking reagent (Cat# 101,302, BioLegend, San Diego, USA) for 10 min. Thereafter, cells were incubated with following antibodies; anti-P-cadherin (Cat# AF761, R&D Systems, Minnesota, USA), anti-CD49f-APC (Cat# 17–0495-82, Invitrogen, Frankfurt, Germany), anti-CD45-Vio-Blue (Cat# 130–092-910, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD-90.2-PE (Cat# 130–102-489, Miltenyi, Bergisch Gladbach, Germany), and CD31 (Cat#DIA 310, clone: SZ31, Dianova, Hamburg, Germany). An Aria III cell sorter (BD Biosciences, Heidelberg, Germany) was used to sort the hair matrix cells (PCAD + CD49f + CD45-CD90.2- CD31-CD326-).

The hindguts were explanted from E10.5 embryos. The gonads exhibiting Oct4-EGFP fluorescence were pooled and then dissociated into single cells by incubation with 0.05% Trypsin-EDTA in phosphate-buffered saline (PBS) at 37 °C for 10 min. When the cell suspensions were washed once in DMEM/F12 containing 10% FBS, and the cell clumps and tissue debris were removed by filtering, the cell suspensions were passed through a cell strainer (Falcon, Fisher Scientific, Thermofisher scientific, Taipei, Taiwan). Afterwards, the EGFP⁺ PGCs were directly sorted and collected into the PGC medium using fluorescence-activated cell sorting (FACS) by the AriaIII cell sorter (BD Biosciences, Stockholm, Sweden). The sorted PGCs were cultured in DMEM containing 10% FBS, 100 U/mL penicillin/streptomycin, and supplemented with growth factors (50 ng/mL soluble SCF from RD Systems Minneapolis; and 10 ng/mL LIF from Chemicon International). In the experiments in Figures 4 to 7, the PGCs were isolated by sorting oct4-EGFP⁺ cells from E10.5 embryos.

After informed consent was collected, PBMCs from healthy individuals were isolated from complete buffy coats (Sanquin) using Ficoll gradient centrifugation. pHLA tetramer-positive T cells were isolated from 250–750 × 10⁶ PBMCs from donors negative for the HLA of interest, as previously described.¹² (link) In brief, PBMCs were stained with PE-labeled pHLA tetramers for 10 min at 37°C or 1 h at 4°C. Subsequently, MACS was performed using anti-PE Microbeads (Miltenyi Biotec). The positive fraction was stained with Alexa Fluor700-conjugated anti-CD8 (catalog MHCD0829) (Invitrogen), FITC-conjugated anti-CD4 (catalog 555346) (BD Pharmingen), anti-CD14 (catalog 555397) (BD Pharmingen), and anti-CD19 (catalog 555412) (BD Biosciences). pHLA tetramer-positive CD8⁺ T cells were single-cell sorted using an Aria III cell sorter (BD Biosciences) in 96-well round-bottom culture plates (Greiner Bio-one) containing irradiated PBMCs (50,000 cells/well) (3500RAD) and EBV-LCLs (5000 cells/well) (5000RAD) in 100 μL TCM supplemented with 0.8 μg/mL phytohemagglutinin (PHA) (Thermo Fisher Scientific). Expanding T cell clones were used in functional assays 7–14 days after stimulation. pHLA tetramer staining was performed using PE-conjugated pHLA tetramers for 15 min at 37°C and fluorescence was measured on an LSRII (BD Biosciences) and analyzed using Diva (BD Biosciences) or FlowJo software (TreeStar).

For cell surface staining, cell suspensions were labeled with Abs in PBS, 1% FCS, 2 mM EDTA. For intracellular staining, cells were fixed and permeabilized using Foxp3 staining buffer kit (BD Biosciences) or the BD cytofix/cytoperm kit. For pERK staining, cells were fixed in 2% paraformaldehyde, washed and permeabilized in ice-cold methanol for 30 min, washed twice in PBS 10% FCS, and stained for 1 h. Samples were run on LSR-IIB or Fortessa II (BD Biosciences) and analyzed with FlowJo software (Free Star). An Aria III cell sorter (BD Biosciences) was used to isolate naive CD8 T cells at >97% purity as judged by cell surface marker expression. Coulter CC Size standard beads (Beckman Coulter) were used for calculating cell numbers. Abs were from eBioscience: CD8 (53-6.7), CD44 (IM7), CD122 (5H4), CXCR3 (173), CCR7 (4B12), NKG2D (CX5), CD45.1 (A20), Eomes (Dan11mag), T-bet (4B10), IL-4 (11B11), IFN-γ (XMG1.2), TNF-α (MP6-XT22), and GranzymeB (NGZB); BD Biosciences: Bcl-2 (3F11), CD4 (RM4-5), αβ TCR (β-chain. H57-597), heat stable Ag (HSA; M1/69), and pan ERK; BioLegend: CCL5 (2E9), PLZF (9E12), and IL-4R (I015F8); Santa Cruz Biotechnology: Egr1 (C19); and Cell Signaling Technology: pERK (p42/p44). mCD1d/PBS57 tetramers were generously supplied by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility.

Ba/F3 cells stably expressing wild-type FLT3 or FLT3 mutations were generated using pMSCV FLT3–WT–GFP and the QuikChange site directed mutagenesis kit XL (Stratagene, Santa Clara, CA, USA). The constructs were confirmed by sequence analysis and transfected into Ba/F3 cells with TurboFect (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were selected for GFP expression using a fluorescence-activated cell sorting analysis (FACS) Aria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA).