Stemspan sfem 2 serum free medium

Peripheral blood (PB) from healthy blood donors (HBDs) and from AL patients were obtained after informed consent. The study was approved by the Medical Ethics Committee of the Canton Geneva (study no. 08-001 and 2020-00176). Diagnosis and clinical and laboratory characteristics of leukemia patients are detailed in Table S1. CD34⁺ cells were enriched using the UltraPure CD34 Microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-100-453) and magnetic-activated cell-sorting separation columns (Miltenyi Biotec, #130-042-201). CD34⁺ cell viability and purity were assessed by flow cytometry after staining cells with CD34-PE-Vio770 (Miltenyi Biotec, clone: AC136, #130-113-180), CD45-VioGreen (Miltenyi Biotec, clone: REA747, 130-110-638), and 7-Amino-Actinomycin D (7-AAD) viability dye for live/dead cell discrimination (Beckman Coulter, Brea, California, CA, USA, #B88526). Samples were analyzed on a Navios 10-color flow cytometer (Beckman Coulter, Brea, CA, USA) and the data interpreted with the Kaluza software (Analysis Version 2.1, Beckman Coulter). CD34⁺ cells were subsequently cultured in StemSpan^TM SFEM II serum-free medium (STEMCELL Technologies, Vancouver, BC, Canada, #09605), supplemented with Flt3L, IL-3, IL-6, SCF and TPO (StemSpan^TM CD34⁺ expansion supplement; STEMCELL Technologies, #02691).

The K562, K562/G01 50 (link), 51 (link), MEG-01 and KU812 CML cell lines used are described in Table S2. K562 KDM6A KO cell lines were generated by CRISPR/Cas9 system according to Zhang's protocol 52 (link). All CML cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 °C in 5% CO2. U2OS cells were obtained from the Hong laboratory (Southeast University, China) and cultured in DMEM supplemented with 10% FBS. Mononuclear cells were isolated from fresh bone marrow isolates using Ficoll-Paque media (GE 17-5442-02) according to the manufacturers recommended protocol before culture in StemSpanTM SFEM II serum-free medium (STEMCELL, 09655, Canada) with addition of four growth factors, 20 ng/mL interleukin 3 [IL-3], 20 ng/mL interleukin 6 [IL-6],100 ng/mL Flt3-ligand and 20 ng/mL granulocyte colony-stimulating factor [G-CSF]( PeproTech).