Infinite m200 nanoquant instrument

Cells were seeded into wells in a 96-well plate (1 × 105 cells/mL, 100 μL per well) to cover a 9 × 6 grid, filling 54 wells. Remaining wells were filled with 200 μL of PBS. After 24 hours, 100 μL volumes of dilutions of nanoparticles in water spanning from 1 nM to 0.01 nM were added to the seeded wells (final concentrations spanning 5 pM to 500 pM). For each functionalized particle, eight dilutions were prepared and for each dilution six replicates were performed. In the remaining 6 wells, 100 μL of PBS was added as a control. Cells were then incubated with complexes for 72 h. After 72 h, 50 μL of a PBS solution of MTT (2.5 mg/mL) was added to each well and then incubated for 3 h. After 3 h, media was aspirated from all wells, leaving purple formazan crystals in those wells with viable cells. To each well, 150 μL of DMSO was added. Plates were then agitated for 10 s and analyzed using a plate reader (NanoQuant Infinite M200 instrument by Tecan Group Ltd.) to determine the absorbance of each well at 570 nm. This reading divided by the average from the reading of the six control wells was plotted to determine the IC50 value of each complex for each cell line.

MSCs obtained from cultures of sarcoma and healthy donors were harvested at early and late passages and washed in PBS buffer. DNA was isolated using the Nucleospin Blood kit (Macherey-Nagel, Dueren, Germany) according to manufacturer’s instructions. DNA quality and quantity were assessed with a NanoQuant Infinite M200 instrument (Tecan Group Ltd, Männedorf, Switzerland) before sequencing.
DNA samples were analyzed for mutational screening of TP53, CDKN1A/p21 and MDM2 genes. The 11 exons of TP53, the 3 exons of CDKN1A along with exon–intron junctions, and SNP309 (rs2279744) in MDM2 were PCR-amplified using primer sequences that will be available upon request. The amplification products were purified using ExoSap-IT reagent (USB Corp., Cleveland, OH, USA) and sequenced in both the forward and reverse directions using BigDye Terminator chemistry version 3.1 (Applied Biosystems, Foster City, CA, USA). Purification of sequencing products was performed with BigDye X-Terminator kit and samples were analyzed using an ABI Prism 3100 automated DNA sequence (Applied Biosystems). Reference sequences for TP53, CDKN1A, and MDM2 were obtained from GenBank (accession numbers NM_000546.4, NM_000389 and NM_002392.3, respectively).

Cell proliferation was measured using the Neutral Red Assay (Sigma-Aldrich, Milan, Italy) as previously reported [83 (link)]. Briefly, after 24 h from seeding melanoma cells (5 × 103 cells/well) onto 96 well plates, cells were transfected with 10 nM mimic or the control (Ctrl). After 72, 96, or 120 h, 10 μL of a neutral red solution (1% acetic acid and 50% ethanol) were added to each well, and cells were incubated for 2 h at 37 °C. Optical density at 540 nm was measured using Infinite^® M200 NanoQuant instrument (Tecan, Salzburg, Austria).