Automax

KOP1 or KOBA cells (10⁶ cells) were co-cultured with OP9 cell monolayers in 10-cm dishes in complete BXH2 medium. After 5 days, overgrown KOBA cells were depleted with gentle pipetting from the culture every 2 days. After 8 days, the cultures were treated with trypsin/EDTA and stained with FITC-anti-CD45 antibody, and then CD45⁻ OP9 cells were isolated using FACSAria II (BD Biosciences) or AutoMax (Miltenyl Biotec, Bergisch Gladbach, Germany). In the OP9 cells co-cultured with KOP1 and KOBA cells were called OP9/P and OP9/L, respectively. For in vitro hematopoiesis, B6 BM cells were stained with a mixture of lineage markers (anti-CD45, anti-Ter119 [Biolegend] anti-CD3, anti-CD4, anti-CD8, anti-Mac-1, anti-Gr-1, anti-B220, and anti-NK.1.1 [BD Biosciences]), and the lin⁻ cells were isolated using AutoMax and cultured on OP9 or OP9/L cell monolayers for 5 days. For the Notch ligand assay, C2C12 cell monolayers were cultured in the absence or presence of KOBA cells for 2 days, and the CD45⁻ C2C12 cells were isolated using AutoMax. K562 or MEG-01 cells (10⁶ cells) were co-cultured with OP9 cell monolayers in 10-cm dishes in RPMI medium. After 8 days, the cultures were treated with trypsin/EDTA, stained with human FITC-anti-CD45RA and APC-anti-CD45RO antibody, and CD45⁻ OP9 cells were isolated using AutoMax (Miltenyl Biotec, Bergisch Gladbach, Germany).