Penicillin streptomycin solution 100x

Three different human PCa cell lines were used: LNCaP (androgen-sensitive adenocarcinoma cells derived from supraclavicular lymph node metastasis) and 22RV1 (carcinoma epithelial cell line derived from androgen-dependent CWR22 xenograft after castration-induced regression and relapse), both purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and PC-3 (androgen-independent cell line originated from a bone metastasis of prostatic adenocarcinoma), that was kindly provided by Dr Ignacio Gil Bazo (CIMA, Pamplona, Spain) as CRPC model. All cell lines were authenticated using STR at the Laboratory of Genetic Identification (Legal Medicine and Toxicology Department) at the University of Granada. The three cell lines were maintained with an RPMI 1640 medium (BioWest) containing 10% Fetal Bovine Serum (FBS), 1% MEM Non-Essential Amino Acids Solution (Gibco), 1% glutaMAX (Gibco) and 1% Penicillin-Streptomycin Solution 100X (BioWest) in a humid atmosphere incubator with 5% CO₂. The cell lines were mycoplasma-free and periodically checked for Mycoplasma by the Cell Culture Unit at GENyO.

Acute Lymphoblastic Leukemia cell line (Nalm-6, NCBI C212) was obtained from the Pasteur Institute collection, Tehran, Iran. Nalm-6 cells were cultured in RPMI-1640 with 2 mM l-glutamine (Gibco™ A1049101) containing 10% fetal bovine serum (FBS) (Gibco™ A3160402), 1% antibiotic (Penicillin–Streptomycin Solution 100X, Biowest, L0022) in a humidified atmosphere of 5% CO₂ incubator at 37 °C. ZME was prepared according to methanolic extract protocol that Saedi Dezaki et al. applicated in their study^{21 (link)}. ZME lyophilized powder was dissolved in dimethylsulfoxide (DMSO) (Merck, CAS 67-68-5) and culture media as main ZME stock, and diverse concentrations of ZME were diluted and obtained from main solution stock. The stock of doxorubicin (EBEWE Pharma, Austria) was also diluted into considered concentration for treatment.