Penicillin streptomycin

The parental MCF-7 (breast cancer), MDA-MB-231 (triple-negative breast cancer), U-87 MG (glioblastoma), A549 (non-small cell lung cancer), and PANC-1 (pancreatic cancer) cell lines and HDFs (human dermal fibroblasts) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 and A549 cells were cultured as an attached monolayer and maintained in RPMI 1640 medium (EuroClone, Milan, Italy) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (EuroClone, Milan, Italy), 1% penicillin-streptomycin (EuroClone, Milan, Italy), and 2 mM L-glutamine. MDA-MB-231 cells were cultured as an attached monolayer and maintained in MEM (EuroClone, Boston, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (EuroClone, Milan, Italy), 1% penicillin-streptomycin (EuroClone, Milan, Italy), and 2 mM L-glutamine. U-87, PANC-1 and HDFs were cultured as an attached monolayer and maintained in DMEM (EuroClone, Milan, Italy) supplemented with 10% (v/v) heat-inactivated FBS (EuroClone, Milan, Italy), 1% penicillin-streptomycin (EuroClone, Milan, Italy), and 2 mM L-glutamine. All cells were incubated at 37 °C in a 5% CO₂ tissue culture incubator (MEMmert, Schwabach, Germany).

B-ALL leukemic cell lines NALM-6 and BV173 were purchased from the American Type Culture Collection and cultured in Roswell Park Memorial Institute (RPMI) 1640 (Euroclone) supplemented with 10% fetal bovine serum (FBS) (Euroclone), 100 IU/mL penicillin/streptomycin (Euroclone) and glutamine (Euroclone). ALL-CM cell line was kindly provided by Professor Fred Falkenburg (Leiden University Medical Center) and kept in culture in X-VIVO (Euroclone) with 3% human serum (Euroclone) and 100 IU/mL penicillin/streptomycin. For in vivo experiments, NALM-6 cells were transduced with a bidirectional lentiviral vector (LV) including the Gaussia luciferase LUCIA (InvivoGen) in sense and the low-affinity nerve growth factor receptor (LNGFR) selection marker in antisense (Lucia+/NGFR+/NALM-6), as previously reported.29 (link) About 293 T cells were used as packaging line for LV production and cultured in IMDM medium (Iscove’s Modified Dulbecco’s Medium) supplemented with 10% FBS, 1% penicillin/streptomycin (100 U/mL, 0,1 mg/mL, Euroclone) and 1% glutamine (2 mM, Euroclone).

The cell line was derived from the fresh tumor obtained from the surgery performed after 3 courses of neoadjuvant chemotherapy. This was established by mechanical disaggregation of the aseptic surgical sample, after three washes in saline solution. Small fragments of tissue were seeded in 24-well plates at high dilution. The culture medium used was DMEM low glucose (Euroclone) supplemented with 20% fetal bovine serum (FBS, Gibco), 2 mmol/L l-glutamine (Euroclone) and 100 g/mL penicillin-streptomycin (Euroclone). Cells were maintained at 37 °C in an atmosphere containing 5% CO₂. When a confluence of spread cells from fragments of tissue was reached, cells were carefully detached and passed in new flasks. Cells were monitored daily to evaluate growth and morphology, and they were carefully detached and re-seeded upon reaching confluence. The RH30 cell line was maintained in RPMI 10% FBS, 2 mmol/L l-glutamine (Euroclone), and 100 g/mL penicillin-streptomycin (Euroclone), whereas the RD18 cell line was cultured in DMEM high glucose 2 mmol/L l-glutamine (Euroclone) and 100 g/mL penicillin-streptomycin (Euroclone). Cell line authentication was achieved by using short tandem repeat (STR) DNA fingerprinting (Eurofins Medigenomix, Ebersberg, Germany), and cells were regularly tested for mycoplasma-free infection.