Biological Sample Preparation
The human osteosarcoma cell line [U-2 OS (ATCC HTB 96)23], derived from a 15-year-old Caucasian female and transfected with a fluorescent reporter cyclin B1 GFP, is selected. The cells are maintained at 37 ºC and 5% CO2 using standard tissue culture techniques. Media used is McCoys 5A modified (Sigma) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 10% fetal calf serum and 1000 mg/ml geneticin.
Cells are treated with 1 µM and 10 µM bolus dose of anticancer agent Topotecan (TPT) (Bailly, 2000). TPT is a water soluble derivative of the alkaloid Camptothecin and act as a topoisomerase I, a nuclear enzyme involved in DNA replication and repair, inhibitor (Wang, 1996). TPT is used for the treatment of a wide range of cancers, including lung (Ichinose et al., 2010), breast (Cheung et al., 2008), ovarian (Lorusso et al., 2010) and bone (Seibel et al., 2007), both in experimental and clinical contexts.
An hour post treatment, the cultured dishes are placed onto a time lapse instrument designed to capture transmission phase images from multi well plates. Image sequences are taken for 115 hours at 15-minute time intervals. The cell lineage data is encoded by ProgeniTRAK (Khan et al., 2006) and is retrieved from a cell lineage database ProgeniDB (Khan et al., 2007).