Synergy 4 plate multileaver

PVCL NGs toxicity was determined on TZM.bl cell line by MTT assay according to the manufacturer’s instructions by using a Synergy 4 Plate Multileaver, Biotek Instrument (USA). A stock suspension of NGs (in order to give 100 µg/mL in the experiment) was prepared by weighing the white solid obtained after freeze-drying and mixing it with nuclease-free water. Then, this sample was sonicated by 15 min to obtain a stable suspension. Lower-concentration suspensions were prepared by dilution of this stock suspension using nuclease-free water. DMSO (10%) and culture medium were used as positive and negative controls of cellular death, respectively. Each experiment was performed in triplicate.

The cytotoxicity of G4-70/30 dendrimer and AMC6 nanoparticle on DCs (7.5 × 104 cells/200 μL) was tested in vitro by MTT assays. MTT assay is a colorimetric assay based on the ability of living cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to form soluble formazan crystals. This assay was carried out according to the manufacturer’s instructions (MTT, Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells were treated with increasing concentrations of the dendrimers and, after 24, 48, or 72 h of incubation, the supernatant containing the dendrimer was removed and replaced with 200 μL of Opti-MEM^® (Gibco, London UK) containing 0.5 mg/mL of MTT substrate. After 2 h of incubation under cell culture conditions, the plate was centrifuged at 2000 rpm for 10 min, and the supernatants were discarded. The formazan crystals were diluted in 200 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, St-Louis, MO, USA). The plate was shaken at 700 rpm and formazan concentrations were determined by spectrophotometry (Synergy 4 Plate Multileaver, Biotek Instrument, Winooski, USA) at a wavelength of 570/690 nm. The spectrophotometer was calibrated at 0 using only Opti-MEM^®. Absorbance values were interpreted as a measurement of cell viability. DMSO was used as a positive control of cell death. Each experiment was performed by triplicate.