Trypsin

The S2–013 cell line is a clonal derivative of the Suit2 cell line and was a kind gift from the Tony Hollingsworth laboratory at the University of Nebraska Medical Center. The MiaPaCa2 IKK2-KO and parental wild-type MiaPaCa2 cell lines were a kind gift from the Amar Natarajan laboratory at the University of Nebraska Medical Center. All other cell lines in this study were obtained from American Type Culture Collection (Manassas, VA, USA). All human cell lines were authenticated by STR profiling by the Genetics Core at the University of Arizona. Cells were routinely (at the time of initial revival from liquid nitrogen storage and at least every 6 months) determined to be free of mycoplasma contamination by PCR-based methods. Cells were cultured in Dulbecco’s modified Eagle medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 50 IU/mL penicillin, 50 μg/mL streptomycin, and incubated at 37 °C in a humidified incubator with 5% CO₂. Cells were maintained at 10% fetal bovine serum (FBS). Upon reaching 70–80% confluency, cells were passaged by washing with phosphate-buffered saline (PBS) before adding 0.25% trypsin (Caisson Labs, Smithfield, UT, USA) and plating at 25% confluency.

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”).