Cell sorter nir aria 2

Human osteosarcoma cell lines, MG-63 (CRL-1427), Saos-2 (HTB-85) and U-2 OS (HTB-96) were purchased from ATCC (Manassas, Virginia, USA). Briefly, cells were cultured respectively in DMEM-HG (MG-63) or McCoy’s medium (Saos-2, U-2 OS) containing 10% of FBS, 1% GlutaMAX and 50 U/ml penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO₂.
Saos-2-Luc/GFP cell line was generated by transduction with Lentivirus particles containing the CMV promoter for the expression of humanized firefly luciferase (hLUC) and SV40 promoter for the expression of GFP protein according to manufacturer’s protocol (GeneCopoeia). Three days after infection, cells with high levels of GFP expression were selected by Cell Sorter NIR Aria II (BD Bioscience) and expanded for a week in culture medium supplied with Puromycin to generate a stable cell line.

The ID8‐Luc/GFP cell line was generated by transduction with Lentivirus particles containing the CMV promoter for the expression of humanized firefly luciferase (hLUC) and the SV40 promoter for the expression of GFP protein according to manufacturer's protocol (GeneCopoeia). Briefly, ID8 cells were plated at 2 × 10⁴ cells per well (12‐well plate, Corning) and incubated overnight at 37°C in a humidified 5% CO₂ atmosphere. Cells were then infected with 10 MOI of Lenti‐PAC™ plasmid mix (GeneCopoeia Inc.) in the presence of 8 μg/mL polybrene (Sigma). After overnight incubation at 37°C/5% CO₂, the viral supernatant was discarded, and cells were washed with 1× PBS (ThermoFisher) prior to the addition of warmed HG‐DMEM media. Three days after infection, cells with high levels of GFP expression were selected by Cell Sorter NIR Aria II (BD Bioscience) and expanded for a week in HG‐DMEM media in presence of 1 μg/mL puromycin (Invitrogen) to further select transfected cells and generate a stable cell line.