96 well cell culture microplates

For adherent cultures (parental H460 cells), cells were plated in 96-well cell-culture microplates (Costar, USA) at ~2,000 cells per well and incubated overnight in CM. Then, the cells were exposed to the appropriate concentration of drug or vehicle for 72 h. Cell viability for adherent cells was evaluated by the MTT assay. The absorbance of solubilized formazan was read at 570 nm using Gen 5 2.0 All-In-One microplate reader (BioTek Instruments, Inc.).
For floating LTSs, cells growing in Poly-HEMA plates were collected in 15 mL Falcon tubes, centrifuged at 700 rpm × 3 min, and resuspended in fresh SFM. In order to plate the same number of cells, this cell suspension was split in 1 mL aliquots. Vehicle or drugs were added to each aliquot and then 150 μL cell suspension was loaded into each microwell (in a 96-well plate) and incubated for 72 h. For floating LTSs, cell viability was evaluated by the CCK-8 assay (Dojingo Laboratories).
In all cases, the highest concentration of DMSO was used in the control and this concentration was maintained below 0.001% (v/v). This DMSO concentration did not show any significant antiproliferative effect on the cell lines or LTSs in a short-term assay.

Cell viability and proliferation of the canine OS cell lines (Abrams, HMPOS, GD-17 and Gracie) treated with tRNA/miR34-a was determined using a MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) based-colorimetric assay (TACS MTT Cell Proliferation Assay, Trevigen, Gaithersburg, MD), according to manufacturer’s instructions. Briefly, canine OS cells were seeded in 96-well cell culture microplates (Corning Costar, Corning, NY). After 24 hours, cells were treated with tRNA/miR-34a or tRNA/MSA (0, 1, 5, 10, 20 and 30 nM) in triplicate by means of jetPRIME transfection reagent (Polyplus-transfection SA, New York, NY). After 8 hours, transfection mixture was replaced with supplemented RPMI 1640 medium. MTT assays were performed 48 hours after treatment. Cells were incubated with MTT reagent in the dark at 37°C for 2 hours. The detergent reagent was then added to all wells, and plates were returned to the dark at 37°C for other additional 2 hours. Following the incubation, absorbance was measured at 570 and 690 nm employing a microplate spectrophotometer (SpectraMax 190, Molecular Devices LLC., Sunnyvale, CA). IC₅₀ and Hill Slope values were calculated using the non-linear regression inhibitory concentration versus normalized analysis with variable slope implemented in GraphPad Prism v7.04 (GraphPad Software, La Jolla, CA).