Mycoplasma pcr

The human SH-SY5Y cell line has been purchased from ECACC and cultivated in Dulbecco’s minimum essential medium, completed with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Corning, USA) at 37°C in 95% O₂ and 5% CO₂ incubator (Thermo, USA). For the PD in vitro model, the SH-SY5Y cell line was differentiated with all trans-retinoic acid (10mM) and 3 days with phorbol (80nM). At 6 DIV, cells were treated with SLAB51 0.1 mg/ml of extract for 2 hours and then added 6-OHDA (Sigma Aldrich, USA) (35μM) for 24 hours. All experiments were performed at 19^th passage and the cell culture were tested to Mycoplasma presence (Mycoplasma PCR, abm, USA).

As cellular model, human neuroblastoma cell line SH-SY5Y was used (ATCC # CRL-2266) and cultured following manufacturer’s protocols in Dulbecco’s minimum essential medium (DMEM) complemented with 10% heat-inactivated fetal bovine serum (FBS), and 1% L-glutamine at 37 °C (95% air and 5% CO2). All experiments were made at the same passage and the cell culture was routinely tested for Mycoplasma (Mycoplasma PCR, ABM, USA).
For obtaining dopaminergic-like phenotype, cells were cultured under differentiation conditions. 24 h after plating (1.5 × 10⁴ cells/cm²), cells were grown in culture media supplemented with 1% FBS and 10µM All-Trans Retinoic acid (ATRA) for three days. The media was then removed and replaced with culture medium supplemented with 1% FBS and 80nM 12-O-tetrade-canoyl-phorbol-13-acetate (TPA) and maintained in these culture conditions for another three days. For the setup of PD model, 6-OHDA was added in the dopaminergic-like differentiated cells (see the following paragraph).