Mycoplasma detection kit

The human embryonic kidney epithelial cell line HEK293T (ATCC, CRL-3216), and human lung epithelial cell line A549 (ATCC, CCL-185) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; high glucose, Sigma) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) PenStrep (Sigma) and maintained in a 5% CO₂ incubator at 37°C. THP1 (ATCC, CRL-TIB-202) cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (R8758, Sigma) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) PenStrep (Sigma) and maintained in a 5% CO₂ incubator at 37°C. Each cell lines were confirmed free from mycoplasma contamination by testing with mycoplasma detection kit (Sigma).

The human bone marrow–derived mesenchymal stromal cell line HS5 was obtained from the ATCC and was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. PC-3ML-Luc cells were obtained from M.G. Pomper (Johns Hopkins Medical Institutions, Baltimore, MD) and maintained as previously described (39 (link)). Murine prostate cancer cell line RM1 was provided by T.C. Thompson (Baylor College of Medicine, Houston, TX), and RM1-BM cells (derived from C57BL/6 J mouse bone metastases) were maintained in DMEM supplemented with 10% fetal bovine serum as previously described (34 (link)). Stable MDA-9/Syntenin knock-out clones were generated using sgRNA/Cas9 all-in-one expression clone targeting SDCBP (mda-9/syntenin) (Cat No: HCP216662-CG01-1) from GeneCopoeia. At 48 h posttransfection, cells were incubated with 5 mg/mL Neomycin to select resistant clones, and lack of MDA-9/Syntenin expression was confirmed by western blotting analysis. All cell lines were regularly monitored (once every 3 mo) for mycoplasma contamination using a mycoplasma detection kit (Sigma). Bone marrow stromal cells (BM-MSCs) were isolated from WT and mda-9^−/− mice according to a previously described protocol (75 ).