Pcr mycoplasma detection set

Prostate cell lines, LNCaP, 22RV1, DU145, PC-3 (malignant), and RWPE (benign) were used for in vitro studies. LNCaP and 22Rv1 cells were grown in RPMI 1640, whereas DU145 and PC-3 cells were maintained in MEM and 50% RPMI-50% F-12 medium, while RWPE was cultured in Keratinocyte-SFM, containing human recombinant Epidermal Growth Factor 1-53 and Bovine Pituitary Extract (GIBCO, Invitrogen, Carlsbad, CA, USA), respectively. HEK293Ta were maintained in DMEM. All basal culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (GIBCO, Invitrogen, Carlsbad, CA, USA). Cells were maintained in an incubator at 37 °C with 5% CO₂. All cell lines were G-banding karyotyped (for validation) and routinely tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set, Clontech Laboratories).
One micromolar of the DNA methyltransferases inhibitor 5-aza-2-deoxycytidine (5-Aza-CdR; Sigma-Aldrich, Schnelldorf, Germany) was used for DNA demethylation. Cells were harvested and RNA extracted after 72-h exposure to the demethylating agent.

The BC cell lines 5637 and J82 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Both cell lines were derived from transitional cell carcinoma of the human urinary bladder, with 5637 being classified as grade II and J82 as grade III (stage pT3) [20 (link)]. BC cell lines were cultured in MEM to ensure optimal cell growth and maintenance of epithelial cell characteristics. The culture medium was prepared as indicated by the manufacturer and supplemented with 10% FBS and 100 units·mL⁻¹ penicillin/100 μg·mL⁻¹ streptomycin. All cell lines were routinely tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set, Clontech Laboratories).

PCa cell lines LNCaP and PC-3 were kindly provided by Prof. Ragnhild A. Lothe from the Department of Cancer Prevention at the Institute for Cancer Research, Oslo, Norway, whereas DU145 was kindly provided by Professor Fátima Baltazar at ICVS, University of Minho, Braga, Portugal and 22Rv1 cells were kindly provided by Dr. David Sidransky at the Johns Hopkins University School of Medicine, Baltimore, MD, USA. LNCaP and 22Rv1 cells were grown in RPMI 1640, DU145 cells were maintained in MEM and PC-3 cells were grown in 50% RPMI-50% F-12 medium (GIBCO, Invitrogen, Carlsbad, CA, USA). All basal culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (GIBCO, Invitrogen, Carlsbad, CA, USA). Cells were maintained in an incubator at 37ºC with 5% CO₂. All PCa cell lines were karyotyped by G-banding (for validation purposes) and routinely tested for Mycoplasma spp. contamination (PCR Mycoplasma Detection Set, Clontech Laboratories). Hydralazine was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS (GIBCO, Invitrogen, Carlsbad, CA, USA). For control purposes, cell lines were exposed to the vehicle of the drug (PBS).