Establishment and Authentication of Human Osteosarcoma Hu09 Cells

Human osteosarcoma Hu09 cells (CVCL-01298) were obtained from Dr. Lin Ren at the national cancer institute (NCI) as previously reported^{30 (link)}. Cells were STR analyzed and authenticated. Cells were cultured in RPMI 1640 medium (Caisson Labs, Smithfield, UT) supplemented with 10% fetal bovine serum (Corning, Smithfield, UT) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO), and incubated at 37 °C with 5% CO₂ in a humidified incubator. Cells received frequent media changes and were expanded to 80% confluency before being detached with 0.25% trypsin-ethylenediaminetetraacetic acid (0.25% trypsin-EDTA, 1X, phenol red, Caisson Labs, Smithfield, UT) for experiments or sub-culturing at a lower concentration. Mycoplasma tests were carried out to monitor potential cell culture contaminations. The cultured Hu09 cells were then subjected to immunofluorescence staining (see section “Immunofluorescence staining”) for establishing the imaging thresholds for CTC detection (see section “Fluorescence imaging and CTC enumeration”).