Facs ariaiii

To isolate the cell population with an ALDH activity, an ALDEFLUOR assay kit (STEMCELL Technologies) was used according to the manufacturer. Allophycocyanin (APC) mouse anti-human CD117 and CD133 (BD Biosciences, San Diego, CA, USA) were used to isolate CD117⁺ and CD133⁺ cells. After trypsinization, cells were washed with 1 mL cold PBS by centrifuging at 500× g for 5 min. Cells were suspended in PBS and incubated with anti-CD117 and anti-CD133 antibodies. After incubation for 30 min on ice in the dark, cells were washed twice with PBS, and resuspend in PBS. The CD117⁺ and CD133⁺ cells were isolated by using a Flow cytometry sorter (BD FACS Aria Ⅲ, San Diego, CA, USA).

Blood samples from healthy donors were obtained from the Australian Red Cross Blood Service. Peripheral blood mononuclear cells were prepared by Ficoll Histopaque (GE Healthcare, Chicago, IL, USA) and cells were cryopreserved in 50% fetal bovine serum, 40% RPMI (Thermo Fisher Scientific, Boston, MA, USA) and 10% dimethyl sulfoxide for 10–100 million/tube. Nearly 50–100 million peripheral blood mononuclear cells were thawed and washed in RPMI medium. Cells were then incubated with Fc block (BioLegend, San Diego, CA, USA) for 30 min at 4°C. After washing, cells were stained with antibodies against CD19 (BV421, HIB19; BioLegend), IgD (PerCP‐CY5.5, IA6‐2; BD, San Diego, CA, USA), CD27 (PE‐CY7, M‐T271; BD), CD10 (PE, HI10a; BD), IgM (BV421, MHM‐88; BioLegend), CD3 (APC‐CY7, SK7; BD), CD14 (APC‐CY7, MOP9; BD) and viability dye (eFluor780; eBioscience). Transitional B cells (CD19⁺CD27⁻IgD⁺CD10⁺) with IgM^hi (top 25%) and IgM^lo (bottom 25%), and mature naïve B cells (CD19⁺CD27⁻IgD⁺CD10⁺) with IgM^hi (top 25%) and IgM^lo (bottom 25%) were sorted on BD FACS Aria Ⅲ into 350 µL RLT lysis buffer (Qiagen, Venlo, Netherlands) and frozen at –80°C. For each donor, 10 000 cells were sorted for each population.