Human cd133 microbead kit

hUCB samples (60–80 ml/unit) were obtained from healthy full-term births with parental informed consent at the Southwest Hospital, Third Military Medical University (Army Medical University). Fresh umbilical cord blood cells were separated within 12 h and used in subsequent experiments. Human mononuclear cells were isolated by density-gradient centrifugation with Ficoll-Paque PREMIUM 1.077 g/ml (GE Healthcare, Little Chalfont, United Kingdom). Red blood cells were removed with red cell lysate. According to the manufacturer’s instructions, CD133⁺ cells were isolated from human monocyte cells with a human-CD133 MicroBead Kit (130-100-830, Miltenyi Biotec) by magnetic bead separation. To assess the sorting, CD133⁺ cells were stained by primary PE-conjugated CD133 antibody (130-113-670, Miltenyi Biotec). The cell purity was 95%. CD133⁺ cells were seeded at 2 × 10⁵ cells/well in 24-well plates (NEST, China) and then cultured in StemMACS™ HSC Expansion Media, human (130-100-463, Miltenyi Biotec) at 37°C in a humidified atmosphere containing 5% CO₂ for 7 days, then PE-conjugated anti-human-CD133 (130-113-670, Miltenyi Biotec) and APC-conjugated anti-human-CD34 (130-113-176, Miltenyi Biotec) antibodies were used to obtain CD133⁺CD34⁺ cells by flow sorting.

The details for staining CD133, Nanog, and p-VEGFR2 are shown in the supplementary data. A human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133⁺ cells from HepG2 cells and pHCCs. These CD133⁺ cells were used to detect sphere formation. The details are provided in the supplementary data.